NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis

Abstract

Reactive oxygen species (ROS) have been proposed to function as second messengers in abscisic acid (ABA) signaling in guard cells. However, the question whether ROS production is indeed required for ABA signal transduction in vivo has not yet been addressed, and the molecular mechanisms mediating ROS production during ABA signaling remain unknown. Here, we report identification of two partially redundant Arabidopsis guard cell-expressed NADPH oxidase catalytic subunit genes, AtrbohD and AtrbohF, in which gene disruption impairs ABA signaling. atrbohD/F double mutations impair ABA-induced stomatal closing, ABA promotion of ROS production, ABA-induced cytosolic Ca(2+) increases and ABA- activation of plasma membrane Ca(2+)-permeable channels in guard cells. Exogenous H(2)O(2) rescues both Ca(2+) channel activation and stomatal closing in atrbohD/F. ABA inhibition of seed germination and root elongation are impaired in atrbohD/F, suggesting more general roles for ROS and NADPH oxidases in ABA signaling. These data provide direct molecular genetic and cell biological evidence that ROS are rate-limiting second messengers in ABA signaling, and that the AtrbohD and AtrbohF NADPH oxidases function in guard cell ABA signal transduction.

Fig. 1. AtrbohD and AtrbohF genes are upregulated by ABA in guard cells. (A) Genechip experiments show expression levels of AtrbohD and AtrbohF mRNA in guard cells and mesophyll cells. Gene chip experiments were performed with guard cell and mesophyll cell RNA that was extracted from WT plants sprayed with 100 µM ABA or water 4 h prior to RNA isolation. (B) AtrbohD promoter drives GUS expression in guard cells of WT plants expressing the AtrbohD::GUS fusion construct. (C) WT plants were sprayed with 100 µM ABA for 15, 30, 60 or 120 min prior to RNA isolation. Total RNA (25 µg) extracted from rosette leaves was separated in a denaturing gel and then transferred onto a nylon membrane. The blot was hybridized with ³²P-labeled AtrbohD or AtrbohF cDNA. The same blot was probed with ³²P-labeled 18S rDNA to show relative amounts of RNA samples.

Fig. 2. ABA-induced stomatal closing and ABA-induced ROS generation are disrupted in the atrbohD/F double mutant. (A) Stomatal aperture measurements show that ABA-induced stomatal closing is partially reduced in atrbohF and atrbohD/F double mutants. (B) ABA (50 µM) induces ROS increases in guard cells of WT (three experiments; n = 44 guard cells before ABA treatment, n = 41 guard cells after ABA treatment). ABA failed to induce an increase in ROS levels in guard cells of atrbohD/F double mutant (three experiments; n = 59 guard cells before ABA treatment, n = 39 guard cells after ABA treatment). In (A), n = 6 experiments (120 stomatal apertures per data point) for WT; n = four experiments (80 stomatal apertures) for atrbohD/F; n = 3 experiments each (60 stomatal apertures each) for atrbohD and for atrbohF. Starting stomatal apertures were: 3.46 ± 0.63 µm (WT), 3.88 ± 0.37 µm (atrbohD), 3.36 ± 0.48 µm (atrbohF), 3.16 ± 0.44 µm (atrbohD/F). Error bars represent ± SEM relative to number of experiments. Error bars are smaller than symbols when not visible.

Fig. 3. Exogenous H₂O₂ rescues stomatal closing in atrbohD/F double mutant. H₂O₂ induces stomatal closing both in atrbohD/F and WT. Stomatal apertures were measured 3 h after addition of 100 or 500 µM H₂O₂. n = 3 experiments (60 stomatal apertures) for WT; n = 3 experiments (60 stomatal apertures) for atrbohD/F. Starting stomatal apertures: 3.81 ± 0.20 µm (WT), 3.40 ± 0.04 µm (atrbohD/F). Error bars represent ± SEM relative to three independent experiments. Error bars are smaller than symbols when not visible.

Fig. 4. atrbohD/F mutation impairs ABA-induced [Ca²⁺]_cyt elevations in guard cells. (A) Fluorescence emission ratio (535/480 nm) shows responsiveness of ABA-induced [Ca²⁺]_cyt elevations at 5 µM ABA in WT guard cells. Thirty of 35 cells (85.7%) showed ABA-mediated [Ca²⁺]_cyt elevations whereas five of 35 cells (14.3%) showed no response. (B) Fluorescence emission ratio (535/480 nm) shows examples of ABA-induced [Ca²⁺]_cyt elevations at 5 µM ABA in atrbohD/F guard cells (15 of 33 cells = 45.5%). Traces showing ABA-induced [Ca²⁺]_cyt transients are shown in the top panels of (A) and (B), and those with no clear ABA-induced [Ca²⁺]_cyt elevations are shown in the bottom panels of (A) and (B). Vertical arrow lines indicate when guard cells were challenged with 5 µM ABA. [Ca²⁺]_cyt transients were counted when changes in [Ca²⁺]_cyt ratios were ≥0.1 U. (C) Stack column representation of number of ABA-induced [Ca²⁺]_cyt transients recorded in WT (n = 35) and atrbohD/F (n = 33) guard cells at 5 µM ABA.

Fig. 5. ABA-activation of I_Ca channels is abolished in atrbohD/F guard cells. (A and B) ABA (50 µM) activated I_Ca channel currents in WT guard cells. Current traces before and after ABA activation of I_Ca channels are shown in a representative cell in (A) and average current– voltage curves of 18 cells are shown in (B). (C and D) ABA failed to activate I_Ca channel currents in atrbohD/F guard cells. A response in a representative cell is shown in (C) and average current–voltage curves (n = 10 cells) are shown in (D). Symbols of the atrbohD/F guard cells perfused with ABA overlap with atrbohD/F control guard cell symbols when not visible.

Fig. 6. ROS activate plasma membrane I_Ca channels both in atrbohD/F and WT guard cells. (A and C) H₂O₂ activation of I_Ca channels in a representative cell of WT and atrbohD/F, respectively. (B and D) Average current–voltage curves (WT, n = 10; atrbohD/F, n = 7). Error bars represent SEM. Error bars are smaller than symbols when not visible.

Fig. 7. H₂O₂- and ABA-activated I_Ca currents are Na⁺ permeable in guard cells. (A) Whole-cell current recordings without (no ABA) and with 50 µM ABA (+ ABA) in the same guard cell bathed in 200 mM NaCl. (B) Average Na⁺ currents at –196 mV show that both ABA and H₂O₂ activated inward Na⁺ currents in Arabidopsis guard cells (H₂O₂, n = 7 cells; ABA, n = 6 cells).

Fig. 8. Root growth and seed germination in atrbohD/F show reduced ABA sensitivity. (A) Seven-day-old WT, atrbohD, atrbohF and atrbohD/F seedlings were placed on MS medium supplemented with 0 and 10 µM ABA. Root elongation was measured after 5 days. Each data point represents the mean value of 12–15 seedlings. Error bars represent ±SEM. (B) The atrbohD/F mutant shows partial reduction in ABA sensitivity of seed germination inhibition. Error bars are smaller than symbols when not visible. Error bars represent ±SE of n = 3 independent experiments; >360 seeds at each data point.