Cytoplasmic alkalization precedes reactive oxygen species production during methyl jasmonate- and abscisic acid-induced stomatal closure

Abstract

Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis wild type, ABA-insensitive (ost1-2), and MJ-insensitive mutants (jar1-1) in order to examine a crosstalk between ABA and MJ signal transduction. Some of the experiments were performed on epidermal strips of Pisum sativum. Stomata of jar1-1 mutant plants are insensitive to MJ but are able to close in response to ABA. However, their sensitivity to ABA is less than that of wild-type plants. Reciprocally, the stomata of ost1-2 are insensitive to ABA but are able to close in response to MJ to a lesser extent compared to wild-type plants. Both MJ and ABA promote H(2)O(2) production in wild-type guard cells, while exogenous application of diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, results in the suppression of ABA- and MJ-induced stomatal closure. ABA elevates H(2)O(2) production in wild-type and jar1-1 guard cells but not in ost1-2, whereas MJ induces H(2)O(2) production in both wild-type and ost1-2 guard cells, but not in jar1-1. MJ-induced stomatal closing is suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Furthermore, MJ induces alkalization in guard cell cytosol, and MJ-induced stomatal closing is inhibited by butyrate. Analyses of the kinetics of cytosolic pH changes and reactive oxygen species (ROS) production show that the alkalization of cytoplasm precedes ROS production during the stomatal response to both ABA and MJ. Our results further indicate that JAR1, as OST1, functions upstream of ROS produced by NAD(P)H oxidases and that the cytoplasmic alkalization precedes ROS production during MJ or ABA signal transduction in guard cells.

Figure 1.

Dose-response curves of ABA- (squares) and MJ- (circles) induced stomatal closure in Landsberg erecta (A), jar1-1 mutant (B), and ost1-2 (C) mutant plants of Arabidopsis, respectively. The stomata of abaxial epidermis from leaves were allowed to open in light for 2 h, then ABA or MJ was applied for 2 h. Results are the averages ± se (n = 60) from at least 3 independent experiments.

Figure 2.

ABA- or MJ-induced stomatal closing (A) in the presence of the protein kinase inhibitors K252a (B), ML7 (C), and the calmodulin antagonist W7 (D). Stomata of leaf epidermis were allowed to open in light for 2 h, and then incubated for 2 h in ABA or MJ. K252a, ML7, and W7 were added 30 min before the addition of ABA or MJ. Results are the averages ± se (n = 60) from at least 3 independent experiments.

Figure 3.

Dose-response curves of ABA-induced (squares) and MJ-induced (circles) stomatal closure in atrbohD/F double mutant plants and gork1 mutant plants. Stomata of leaf epidermis were allowed to open for 2 h under light, then ABA or MJ was applied for 2 h. Results are the average ± se (n = 60) of 3 to 4 independent experiments.

Figure 4.

Change in the pH of the external solution after permeation of the guard cell plasma membrane by digitonin in control protoplasts (A), ABA-treated protoplasts (B), or MJ-treated protoplasts (C). Guard cell protoplasts were incubated in the presence of 20 μm ABA or MJ for 30 min before application of digitonin. Values indicate the external solution pH at time of application of digitonin; 10 nmol of HCl were injected into the medium at the end of each experiment for calibration.

Figure 5.

Kinetics of pH change (A) and ROS production (B) in guard cells in response to 20 μm ABA or 20 μm MJ; pH changes (A) were determined using the null-point method and H₂O₂ production (B) using the fluorescent dye H₂DCF-DA as described in “Materials and Methods”. Each data point is the mean ± se from at least 3 independent experiments.

Figure 6.

The pH changes and ROS production in guard cells of P. sativum. Changes in pH (A) or ROS (B) were monitored by using BCECF-AM or H₂DCF-DA after the addition of ABA (squares) and MJ (circles). The pixel intensities of fluorescence at each given point were determined and the relative changes in pH or ROS production were expressed by considering solvent control at zero time as the standard (100%). Each data point is the mean ± se from at least 3 independent experiments. Note the different time scales in A and B.

Figure 7.

MJ- or ABA-induced H₂O₂ production in guard cells is inhibited by 0.5 mm butyrate. Photographs were taken from a representative lot of guard cells from epidermal strips loaded with H₂DCF-DA, untreated (A) or submitted to a 30-min pretreatment with 20 μm MJ (B), or 20 μm ABA (E). C and F, Effects of 0.5 mm butyrate on MJ- or ABA-induced H₂O₂ production, respectively. Photographs were taken using fluorescence (A–C, E, F) or light microscopy (D); bars represent 10 μm.

Figure 8.

Model for the sequence of events in the MJ signaling cascade leading to stomatal closure. This linear model integrates our results from the different mutants and the use of inhibitors. Ca²⁺-CaM PK, calcium-calmodulin activated protein kinase; Ica, calcium influx at the plasma membrane. abi1 and abi2 have been placed according to Murata et al. (2001) and Mustilli et al. (2002).