Sulfate is Incorporated into Cysteine to Trigger ABA Production and Stomatal Closure

Abstract

Plants close stomata when root water availability becomes limiting. Recent studies have demonstrated that soil-drying induces root-to-shoot sulfate transport via the xylem and that sulfate closes stomata. Here we provide evidence for a physiologically relevant signaling pathway that underlies sulfate-induced stomatal closure in Arabidopsis (Arabidopsis thaliana). We uncovered that, in the guard cells, sulfate activates NADPH oxidases to produce reactive oxygen species (ROS) and that this ROS induction is essential for sulfate-induced stomata closure. In line with the function of ROS as the second-messenger of abscisic acid (ABA) signaling, sulfate does not induce ROS in the ABA-synthesis mutant, aba3-1, and sulfate-induced ROS were ineffective at closing stomata in the ABA-insensitive mutant abi2-1 and a SLOW ANION CHANNEL1 loss-of-function mutant. We provided direct evidence for sulfate-induced accumulation of ABA in the cytosol of guard cells by application of the ABAleon2.1 ABA sensor, the ABA signaling reporter ProRAB18:GFP, and quantification of endogenous ABA marker genes. In concordance with previous studies, showing that ABA DEFICIENT3 uses Cys as the substrate for activation of the ABSCISIC ALDEHYDE OXIDASE3 (AAO3) enzyme catalyzing the last step of ABA production, we demonstrated that assimilation of sulfate into Cys is necessary for sulfate-induced stomatal closure and that sulfate-feeding or Cys-feeding induces transcription of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, limiting the synthesis of the AAO3 substrate. Consequently, Cys synthesis-depleted mutants are sensitive to soil-drying due to enhanced water loss. Our data demonstrate that sulfate is incorporated into Cys and tunes ABA biosynthesis in leaves, promoting stomatal closure, and that this mechanism contributes to the physiological water limitation response.

Figure 1.

Sulfate Induces Closure of Arabidopsis Stomata in a Dose- and Time-Dependent Manner and by Activation of NADPH Oxidases. (A) Stomatal apertures of epidermal peels from 5-week-old soil-grown Arabidopsis wild-type plants incubated for 180 min with water containing up to 20 mM MgSO₄. The apertures of 50 stomata were determined from peels of five individual plants (n = 5). Images illustrate typical stomatal apertures in response to the treatments. The applied sulfate concentration (mM) is indicated in white in the photographs. (B) Time course of sulfate-induced stomatal closure in detached leaves fed via the petiole with 2 mM sulfate (white, MgSO₄) or water (black, n = 50, from leaves of five individual plants). (C) Water loss of detached leaves from 5-week-old wild-type plants that were preincubated for 180 min in water (black circles) or 2 mM MgSO₄ (white circles, sulfate). (D) Stomatal apertures of epidermal peels treated with different nutrient salts (15 mM). (E) Quantification of hydrogen peroxide production after fluorescent-labeling with H₂DCF-DA. Epidermal peels were treated with water (control), ABA (50 μM), or sulfate (15 mM) for 180 min before analysis (n ≥ 100). (F) Impact of the selective NADPH-oxidase inhibitor DPI (10 μM, red dash) on ABA-induced and sulfate-induced production of hydrogen peroxide in guard cells of epidermal peels (ABA, 50 μM, sulfate, 15 mM MgSO₄ and DPI, 10 μM, n ≥ 100). (G) Impact of NADPH-oxidase inhibition by DPI on sulfate-induced stomatal closure (n = 50, from peels of five individual plants). Bars represent mean ± sd in (A) to (E) and (G) and mean ± se in (E) and (F). Letters indicate statistically significant differences between groups determined with one-way ANOVA (P < 0.05).

Figure 2.

Sulfate-Induced Stomatal Closure Requires ABA-Signaling Components and ABA-Downstream Effectors. (A) and (C) Impact of ABA (gray, 50 µM) and sulfate (white, 15 mM MgSO₄) on hydrogen peroxide production in guard cells of epidermal peels of five-week-old slac1 (A), and abi2-1 and aba3-1 (C) plants. Data represent mean ± se (n ≥ 100; derived from ≥5 individual plants). (B) and (D) Impact of sulfate (white, 15 mM MgSO₄) on stomatal apertures of the wild-type (WT), slac1 (B) and mutants deficient in ABA sensing (abi2-1, [D]) or ABA production (aba3-1, [D]). Control refers to water. Data represent mean ± sd in (B) and (D) (n ≥ 50, derived from ≥5 individual plants). Letters indicate statistically significant differences between groups determined with one-way ANOVA (P < 0.05).

Figure 3.

Sulfate Triggers ABA Production in Guard Cells in a Concentration-Dependent Manner. (A) The upper panel shows the ABAleon2.1 emission ratio. Signals from guard cells treated with water alone (n = 308), or water supplemented with 2 mM MgSO4 (n = 125), 15 mM MgSO4 (n = 67), or 15 mM MgCl2 (n = 110), respectively. Average ABAleon2.1 emission ratio is calculated per stomatal area. Statistical tests are performed with respect to the water control. The lower panel shows a representative stoma in the given treatment. Letters indicate statistically significant differences between groups determined with one-way ANOVA (P < 0.05). (B) Transcript levels of ABA-responsive genes in sulfate-treated epidermal peels. Epidermal peels were collected from 5-week-old wild-type plants and incubated on water supplemented without (black) or with 2 mM MgSO₄ (white, sulfate) for 3 h. RNA was extracted and the steady-state transcript levels of ABA-marker genes (LEA7, HAl1, RD20, and RD29B) were quantified by RT-qPCR. The transcript levels of respective genes from water-treated samples were set to 1. Data represent mean ± sd (n = 3). Asterisks indicate statistical significant differences as determined with the Student’s t test (*P < 0.05). (C) Impact of petiole-fed ABA or sulfate on the expression of the ABA signaling marker ProRAB18:GFP in detached leaves. Leaves of 25-day-old soil grown ProRAB18:GFP plants were detached and fed via the petiole with ABA (gray, 50 μM) or sulfate (white, 15 mM MgSO₄) dissolved in water (black, Control) for 180 min before quantification of the GFP signal. The upper panel displays a representative image of the epidermis containing guard and pavement cells. Bright field image of the same area is shown for orientation. The lower panel depicts the quantification of GFP-signal intensities in guard- or pavement cells after the treatment. Data represent mean ± se (guard cells: n = 666 for water, n = 534 for sulfate, n = 894 for ABA, from five individual leaves, pavement cells n = 20 regions of interest containing multiple pavement cells for each treatment, from five individual leaves). Letters indicate statistically significant differences between groups determined with one-way ANOVA (P < 0.05).

Figure 4.

Guard-Cell Autonomous ABA Synthesis in the MYB60:ABA3-Complemented aba3-1 Mutant is Sufficient for Sulfate-Induced Stomatal Closure. (A) Impact of ABA (gray, 50 μM) and sulfate (white, 15 mM MgSO₄) on hydrogen peroxide production in guard cells of the MYB60:ABA3 complemented the aba3 mutant that lacks ABA biosynthesis by ABA3 in other cell types than guard cells. (B) Impact of sulfate (white, 15 mM MgSO₄) on stomata closure of wild type, aba3-1, and the MYB60:ABA3 complemented the aba3-1 mutant. Data represent mean ± sd (n ≥ 50 stomata, derived from ≥5 individual plants). Letters indicate statistically significant differences between groups determined with one-way ANOVA (P < 0.05).

Figure 5.

Sulfate-Induced Stomatal Closure Requires Sulfate Reduction and Incorporation of Sulfide into Cys. (A) and (B) Impact of ABA (gray, 50 µM) and sulfate (white, 15 mM MgSO₄) on hydrogen peroxide production in guard cells of sir1-1 (A) and serat tko (B) plants. Data represent mean ± se (n ≥ 100; derived from ≥5 individual plants). (C) Impact of ABA (gray, 50 μM) and sulfate (white, 15 mM MgSO₄) on the stomatal apertures of the wild type (WT) and of mutants with a strongly reduced ability to reduce sulfate to sulfide (sir1-1) or incorporate sulfide into Cys (serat tko). Control refers to water. Data represent mean ± sd in (C) (n ≥ 50 stomata, derived from ≥5 individual plants). Letters indicate statistically significant differences between groups determined with one-way ANOVA (P < 0.05).

Figure 6.

Exogenous Application of Cys Promotes Stomatal Closure in Cys-Synthesis–Limited Mutants. (A) to (C) Impact of sulfate (white, 2 mM MgSO₄), Cys (yellow, 0.5 mM), and Gly (dark gray, 0.5 mM) on the stomatal apertures of wild-type (A), sir1-1 (B), and serat tko (C) plants. Data represent mean ± sd in (n ≥ 50, derived from ≥5 individual plants). Letters indicate statistically significant differences between groups determined with one-way ANOVA (P < 0.05).

Figure 7.

Exogenous Application of Cys Induces ABA Production in Guard Cells and ROS Formation in an ABA3-Dependent Manner. (A) The upper panel shows the ABAleon2.1 emission ratio, calculated from guard cells treated with only water (n = 308), 500 μM Cys (n = 311), 500 μM Gly (n = 54), or 50 μM ABA (n = 91), respectively. The average ABAleon2.1 emission ratio is calculated per stomatal area. Statistical tests are performed with respect to the water control. The lower panel shows a representative stomata subjected to the treatment indicated in the x axis label above. (B) and , (C) Impact of ABA (light gray, 50 μM), Cys (yellow, 0.5 mM), or Gly (dark gray, 0.5 mM) on hydrogen peroxide production in guard cells of wild type (B) and aba3-1 (C) as measured by H₂DCF-DA staining. Data represent mean ± sd in (n = 50, derived from ≥5 individual plants). (D) and (E) Impact of sulfate (white, 15 mM), Cys (yellow, 0.5 mM), Gly (dark gray, 0.5 mM), or ABA (light gray, 50 μM) on transcript levels of NCED3 in leaves of the wild type (D) and stomatal aperture of the wild type and the nced3-2 mutant (E). Data represent mean ± sd (n = 50, derived from ≥5 individual plants for stomatal closure, n = 3 for determination of transcript levels). Letters indicate statistically significant differences between groups determined with one-way ANOVA (P < 0.05).

Figure 8.

Physiological Relevance of Cys-Induced Stomatal Closure. (A) Stomatal aperture in detached leaves of wild type and mutants affected in provision of sulfide for Cys synthesis (sir1-1), synthesis of GSH from Cys (cad2-1), and the sir1-1 cad2-1 double mutant (s1c2). Detached leaves were fed with ABA (gray, 50 μM) or sulfate (white, 15 mM MgSO4) dissolved in water (black, Control) for 180 min before analysis. Data represent mean ± sd (n = 50, derived from five individual plants). Letters indicate statistically significant differences between groups determined with one-way ANOVA (P < 0.05). Please note that feeding of ABA via the petiole can close the stomata in sir1-1 and s1c2. (B) Correlation analysis between endogenous foliar Cys steady-state levels and stomatal aperture in wild type (black), sir1-1 (orange), cad2-1 (red), and s1c2 (blue). The data were dynamically fitted with a linear equation (y = m x +b). The negative slope demonstrates that higher endogenous Cys levels correlate with stomatal closure in mutants with functional ABA biosynthesis and ABA response. The regression coefficient was 0.997 and the coefficient of determination (r²) was 0.995, demonstrating the significant correlation between endogenous Cys steady-state levels and stomatal aperture. (C) and (D) Cys-synthesis–depleted mutants (serat2;1 and oastl-b) are sensitive to soil drying. Five-week-old soil-grown wild type and Cys-synthesis–depleted mutants were subjected to drought stress for 25 d. The serat2;1 and oastl-b mutants suffered from only mild depletion of Cys synthesis capacity and thus grow like the wild type under nonstressed conditions (Heeg et al., 2008; Watanabe et al., 2008). Application of drought resulted in a more pronounced wilting of both Cys-synthesis–depleted mutants when compared with the wild type (C) caused by a significantly greater water loss of both mutants upon soil drying (D). Scale bar = 4 cm. The relative water content of the leaves was determined according to the following equation: (fresh weight – dry weight) / (turgid weight – dry weight). Data represent mean ± se (n = 4 individual plants). Letters indicate statistically significant differences between groups determined with one-way ANOVA (P < 0.05).

Figure 9.

Model for the Function of Sulfate in ABA Biosynthesis and Stomatal Closure. Enzymes catalyzing reactions (black arrows) in the biosynthesis pathways of Cys and ABA as well as the sensing of ABA for stomatal closure are shown in yellow boxes. Red box indicates the nonactive apoenzyme, which requires the cofactor for activation. Asterisks indicate enzymes that have been shown by this study to be essential for sulfate/Cys-induced stomatal closure. The stimulating effects of metabolites or enzymes on downstream reactions are depicted as blue arrows or green open arrows, respectively. Numbers in gray circles indicate references for known regulations/processes not experimentally addressed: 1: Synthesis of Cys is limited by provision of O-actylserine and sulfide (Takahashi et al., 2011). 2: Cys is the substrate of the MoCo-sulfurylase ABA3 required for activation of AAO3 (Bittner et al., 2001). 3: Cys level affects AAO activity in vivo (Cao et al., 2014). 4: PYR/PYL acts as an ABA receptor and controls PP2C activity (e.g. ABI1; Park et al., 2009). 5: PP2C activity regulates activation of OST1 in response to ABA (Vlad et al., 2009). 6: OST1 activates SLAC1 by phosphorylation at multiple residues (Geiger et al., 2009; Lee et al., 2009). 7: OST1 phosphorylates RBOHF (NADPH oxidase; Sirichandra et al., 2009). 8: ROS induce stomatal closure in an ABA2-dependent manner (Sierla et al., 2016). 9: SLAC1 is essential for ABA-induced stomatal closure (Vahisalu et al., 2008).