Elevated CO2-Induced Responses in Stomata Require ABA and ABA Signaling

Abstract

An integral part of global environment change is an increase in the atmospheric concentration of CO2 ([CO2]) [1]. Increased [CO2] reduces leaf stomatal apertures and density of stomata that plays out as reductions in evapotranspiration [2-4]. Surprisingly, given the importance of transpiration to the control of terrestrial water fluxes [5] and plant nutrient acquisition [6], we know comparatively little about the molecular components involved in the intracellular signaling pathways by which [CO2] controls stomatal development and function [7]. Here, we report that elevated [CO2]-induced closure and reductions in stomatal density require the generation of reactive oxygen species (ROS), thereby adding a new common element to these signaling pathways. We also show that the PYR/RCAR family of ABA receptors [8, 9] and ABA itself are required in both responses. Using genetic approaches, we show that ABA in guard cells or their precursors is sufficient to mediate the [CO2]-induced stomatal density response. Taken together, our results suggest that stomatal responses to increased [CO2] operate through the intermediacy of ABA. In the case of [CO2]-induced reductions in stomatal aperture, this occurs by accessing the guard cell ABA signaling pathway. In both [CO2]-mediated responses, our data are consistent with a mechanism in which ABA increases the sensitivity of the system to [CO2] but could also be explained by requirement for a CO2-induced increase in ABA biosynthesis specifically in the guard cell lineage. Furthermore, the dependency of stomatal [CO2] signaling on ABA suggests that the ABA pathway is, in evolutionary terms, likely to be ancestral.

Keywords: ABA receptors; ABA signaling; NADPH oxidases; ROS; Rboh genes; [CO(2)] signaling; guard cells; signaling convergence; stomata; stomatal closure; stomatal density.

Graphical abstract

Figure 1

Stomatal Response to Elevated [CO₂] Requires Generation of Reactive Oxygen Species in Guard Cells (A) Stomatal closure induced by elevated [CO₂] is inhibited by reactive oxygen species (ROS) scavengers Tiron and Tempol. Mean stomatal aperture is significantly reduced in wild-type stomata treated with 700 ppm [CO₂] (ANOVA, p < 0.001) compared with treatment with ambient [CO₂]. This response is disrupted by Tiron or Tempol. Error bars represent SE in this and following figures. (B) Elevated [CO₂] stimulates an increase in guard cell H₂DCFDA fluorescence that is blocked in the presence of Tempol or Tiron. Mean fluorescence was significantly higher in stomata treated with 700 ppm [CO₂] (ANOVA, p < 0.001) compared with treatment with ambient [CO₂] but did not increase when preparations were pretreated with ROS scavengers. Fluorescence expressed as a.u. relative to wild-type value at ambient [CO₂]. (C) Elevated [CO₂]-induced stomatal closure is disrupted in the rbohD rbohF mutant. Mean stomatal aperture is significantly reduced in wild-type stomata treated with 1,000 ppm [CO₂] (ANOVA, p < 0.05) compared with treatment with ambient CO₂, but not in rbohD rbohF stomata. (D) Elevated [CO₂] stimulates an increase in wild-type guard cell H₂DCFDA fluorescence but results in decreased fluorescence in rbohD rbohF guard cells. Mean fluorescence was significantly higher in wild-type treated with 1,000 ppm [CO₂] (ANOVA, p < 0.001) compared with treatment with ambient [CO₂] but decreased in rbohD rbohF guard cells. (E) Representative images showing fluorescence of rbohD rbohF and wild-type guard cells under ambient and elevated (1,000 ppm) [CO₂] as in (D). Insets show representative bright-field images used to determine stomatal apertures from (C). (F) The stomatal density response to elevated (1,000 ppm) [CO₂] requires ROS signaling via NADPH oxidases RbohF and RbohD. Mean stomatal density of wild-type leaves was significantly reduced when grown under 1,000 ppm [CO₂] in comparison to ambient [CO₂] (ANOVA, p < 0.001) but was not reduced in rbohD rbohF at elevated [CO₂].

Figure 2

Stomatal Response to Elevated [CO₂] Requires the PYR/RCAR ABA Receptors (A) Mean stomatal aperture was significantly reduced in wild-type stomata treated with 800 ppm CO₂ (ANOVA, p < 0.001) compared with treatment with ambient CO₂, but this response was disrupted in pyr1 pyl1 pyl4 and pyr1 pyl1 pyl2 pyl4. (B) Exposure to elevated [CO₂] fails to stimulate an increase in guard cell H₂DCFDA fluorescence in ABA receptor mutants. Mean fluorescence was significantly higher in wild-type stomata treated with 800 ppm [CO₂] (ANOVA, p < 0.001) compared with treatment with ambient [CO₂] but did not increase in pyr1 pyl1 pyl4 and pyr1 pyl1 pyl2 pyl4.

Figure 3

Stomatal Aperture Response to Elevated [CO₂] Requires ABA Biosynthesis in Guard Cells (A) Mean stomatal aperture is significantly reduced in wild-type stomata treated with 1,000 ppm CO₂ (ANOVA, p < 0.001), but this response is disrupted in nced3 nced5. (B) Exposure to elevated [CO₂] does not induce a significant reduction in stomatal conductance in nced3 nced5. Mean stomatal conductance was significantly reduced in leaves of wild-type plants exposed to 1,000 ppm [CO₂] (ANOVA, p = 0.0155) compared to ambient [CO₂], but not in nced3 nced5 (ANOVA, p = 0.1615). (C) Exposure to elevated [CO₂] fails to stimulate an increase in guard cell H₂DCFDA fluorescence in nced3 nced5. Mean fluorescence was significantly higher in wild-type stomata treated with 1,000 ppm [CO₂] (ANOVA, p < 0.01) compared with treatment with ambient [CO₂] but did not increase in nced3 nced5.

Figure 4

The Stomatal Density Response to [CO₂] Requires ABA Perception and Biosynthesis (A) Mean stomatal density of wild-type leaves was significantly reduced when grown under 1,000 ppm [CO₂] in comparison to when grown at ambient [CO₂] (ANOVA, p < 0.001) but was not reduced in pyr1 pyl1 pyl2 pyl4 at elevated [CO₂]. (B) Stomatal densities of nced3 nced5 and aba3 were significantly higher than wild-type when grown under either ambient or elevated [CO₂] (1,000 ppm) (ANOVA, p < 0.001) and did not reduce when grown at elevated [CO₂]. Stomatal densities of MYB60_pro::ABA3 or SPCH_pro::NCED3-YFP were not significantly different to wild-type when grown under ambient [CO₂] but reduced significantly when grown at elevated [CO₂] (ANOVA, p < 0.05). See also Figure S1. (C) Tracing of epidermal impressions to illustrate the difference in stomatal densities between wild-type and nced3 nced5 leaves following growth at 1,000 ppm [CO₂]. The scale bar represents 100 μm.