Abscisic acid activation of plasma membrane Ca(2+) channels in guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and downstream of reactive oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C mutants

Abstract

The hormone abscisic acid (ABA) regulates stress responses and developmental processes in plants. Calcium-permeable channels activated by reactive oxygen species (ROS) have been shown recently to function in the ABA signaling network in Arabidopsis guard cells. Here, we report that ABA activation of these I(Ca) Ca(2)+ channels requires the presence of NAD(P)H in the cytosol. The protein phosphatase 2C (PP2C) mutant abi1-1 disrupted ABA activation of I(Ca) channels. Moreover, in abi1-1, ABA did not induce ROS production. Consistent with these findings, in abi1-1, H(2)O(2) activation of I(Ca) channels and H(2)O(2)-induced stomatal closing were not disrupted, suggesting that abi1-1 impairs ABA signaling between ABA reception and ROS production. The abi2-1 mutation, which lies in a distinct PP2C gene, also disrupted ABA activation of I(Ca). However, in contrast to abi1-1, abi2-1 impaired both H(2)O(2) activation of I(Ca) and H(2)O(2)-induced stomatal closing. Furthermore, ABA elicited ROS production in abi2-1. These data suggest a model with the following sequence of events in early ABA signal transduction: ABA, abi1-1, NAD(P)H-dependent ROS production, abi2-1, I(Ca) Ca(2)+ channel activation followed by stomatal closing.

Figure 1.

ABA Activation of Ca²⁺-Permeable I_Ca Currents in Arabidopsis Guard Cell Protoplasts Requires Cytosolic NADPH. (A) and (B) ABA (50 μM) did not activate I_Ca calcium channels when the pipette solution did not include NADPH or NADH. (B) shows two overlapping traces from a guard cell before and after ABA application. (C) and (D) ABA (50 μM) activated I_Ca calcium currents when 5 mM NADPH was added to the pipette solution. (E) and (F) ABA activated I_Ca when 1 mM NADPH was added to the pipette solution. (A), (C), and (E) show average responses, and (B), (D), and (F) show responses in individual cells before and 5 min after ABA application. ABA was added ∼10 min after establishing whole-cell recordings, and whole-cell currents were measured before ABA application and in the same cells 5 min after extracellular ABA application in all recordings. The numbers of cells averaged are given in the text. Closed circles, before ABA addition to batch solution; open circles, 5 min after ABA addition to the same cells. Error bars represent sem.

Figure 2.

ABA Failed to Activate Ca²⁺ Channel Currents in abi1-1 and abi2-1 PP2C Mutant Guard Cells. (A) ABA (50 μM) did not activate I_Ca in abi1-1 guard cells with 5 mM NADPH included in the pipette solution (n = 5). (B) ABA (50 μM) did not activate I_Ca in abi2-1 guard cells with 5 mM NADPH added to the pipette solution (n = 5). Experiments were performed as described in Figure 1. Error bars represent sem.

Figure 3.

H₂O₂ Activates Ca²⁺-Permeable Currents in abi1-1 but Not abi2-1 Arabidopsis Guard Cell Protoplasts. (A) and (B) H₂O₂ (100 μM) activates I_Ca in abi1-1 guard cells (n = 6). (C) and (D) H₂O₂ (100 μM) did not activate I_Ca in abi2-1 guard cells (n = 5). (D) shows two overlapping traces. Experiments were performed as described in Figure 1 except that H₂O₂ was used instead of ABA. Error bars represent sem.

Figure 4.

Differential Effects of H₂O₂ on Stomatal Apertures of abi1-1 and abi2-1. (A) Both ABA (50 μM) and H₂O₂ (100 μM) induced stomatal closing in the wild type (Landsberg erecta ecotype). (B) ABA did not cause stomatal closing, but H₂O₂ elicited partial stomatal closing in abi1-1. (C) Neither ABA nor H₂O₂ elicited stomatal closing of abi2-1. Averages from n = three leaf epidermal experiments are shown (60 stomates per bar). Error bars represent sem.

Figure 5.

Differential Production of ROS in abi1-1 and abi2-1 Guard Cells Treated with ABA. ABA (50 μM) increased ROS production in the wild type (WT) (four experiments, n = 161 cells before ABA treatment, n = 123 cells after ABA treatment) and abi2-1 (nine experiments, n = 221 cells before ABA treatment, n = 207 cells after ABA treatment). ABA did not increase ROS production in abi1-1 (six experiments, n = 110 cells before ABA treatment, n = 150 cells after ABA treatment). Changes in ROS levels were analyzed by measuring H₂DCF fluorescence levels in guard cells in response to ABA or solvent (0.1% ethanol) control applications. Error bars represent sem.

Figure 6.

Model Summarizing the Differential Disruption of ABA Activation of I_Ca Ca²⁺ Channels by the Two abi1-1 and abi2-1 PP2C Mutants. ABA activation of Ca²⁺ channels required cytosolic NAD(P)H, was inhibited by DPI, and was accompanied by ROS production.