RBOHF activates stomatal immunity by modulating both reactive oxygen species and apoplastic pH dynamics in Arabidopsis

Abstract

Stomatal defences are important for plants to prevent pathogen entry and further colonisation of leaves. Apoplastic reactive oxygen species (ROS) generated by NADPH oxidases and apoplastic peroxidases play an important role in activating stomatal closure upon perception of bacteria. However, downstream events, particularly the factors influencing cytosolic hydrogen peroxide (H₂ O₂ ) signatures in guard cells are poorly understood. We used the H₂ O₂ sensor roGFP2-Orp1 and a ROS-specific fluorescein probe to study intracellular oxidative events during stomatal immune response using Arabidopsis mutants involved in the apoplastic ROS burst. Surprisingly, the NADPH oxidase mutant rbohF showed over-oxidation of roGFP2-Orp1 by a pathogen-associated molecular pattern (PAMP) in guard cells. However, stomatal closure was not tightly correlated with high roGFP2-Orp1 oxidation. In contrast, RBOHF was necessary for PAMP-mediated ROS production measured by a fluorescein-based probe in guard cells. Unlike previous reports, the rbohF mutant, but not rbohD, was impaired in PAMP-triggered stomatal closure resulting in defects in stomatal defences against bacteria. Interestingly, RBOHF also participated in PAMP-induced apoplastic alkalinisation. The rbohF mutants were also partly impaired in H₂ O₂ -mediated stomatal closure at 100 μm while higher H₂ O₂ concentration up to 1 mm did not promote stomatal closure in wild-type plants. Our results provide novel insights on the interplay between apoplastic and cytosolic ROS dynamics and highlight the importance of RBOHF in plant immunity.

Keywords: Arabidopsis thaliana; Pseudomonas syringae; reactive oxygen species; redox biosensor; stomatal immunity.

Figure 1

The rbohD mutants are defectives in PAMP‐triggered ROS burst in the apoplast. (a and b) PAMP‐induced apoplastic ROS production detected by luminol assay in Col‐0 WT, rbohD (CS9555) and rbohF (CS9557) mutants (a) and other allelic rbohD (SALK_070610) and rbohF (SALK_034674) mutants (b). The luminescence was measured over time after treatment with control solution or 1 μm flg22 at t = 0 min. Data are means ± SE (n = 6) from a representative experiment.

Figure 2

The H₂O₂ sensor roGFP2‐Orp1 is more oxidised in guard cells of the rbohF mutant. (a) Oxidation state of roGFP2‐Orp1 in Col‐0 WT, rbohD and rbohF guard cells in response flg22. Leaf discs were exposed to control solution or 1 μm flg22 and the ratio 405/488 nm of stomata was quantified at 30 min and 60 min after treatment from images of the fluorescence emission at 517 ± 9 nm following excitation at 488 and 405 nm. Data are means ± SE (n ≥ 50 guard cells) from a representative experiment. Different letters indicate significant differences at P < 0.05 based on a Tukey's HSD test. (b) Correlation between the stomatal aperture and the oxidation state of roGFP2‐Orp1 in guard cells of leaf discs after treatment with control solution or 1 μm flg22 for 30 min and 60 min. The scatter plots show the stomatal aperture as a function of the ratio 405/488 nm for each condition (n ≥ 100). The Pearson correlation coefficient (R) is 0.0651 (P = 0.483) for control treatment and − 0.2428 (P = 0.015) and − 0.2395 (P = 0.011) for flg22 treatment at 30 min and 60 min, respectively. (c) ROS production detected by H₂DCFDA fluorescence in guard cells of Col‐0 WT, rbohD and rbohF epidermal peels 30 min after treatment with control solution or 5 μm flg22. Data are means ± SE (n ≥ 200) from 3 independent experiments. Different letters indicate significant differences at P < 0.05 based on a Tukey's HSD test.

Figure 3

Analysis of the function of RBOHD and RBOHF during stomatal defence responses. (a) Stomatal apertures in WT Col‐0, rbohD and rbohF leaf discs exposed to mock control (10 mm MgCl₂), 10⁸ cfu/ml COR‐deficient Pst DC3000 (Pst COR⁻) bacteria and 5 μm flg22 for 2 h. Data are means ± SE (n ≥ 100) from a representative experiment. (b) Stomatal apertures in Col‐0 WT and other allelic mutants of rbohD (SALK_070610) and rbohF (SALK_034674). Epidermal peels were exposed to control solution or 5 μm flg22 for 2 h. Data are means ± SE (n ≥ 80) from a representative experiment. (c) Bacterial growth in WT Col‐0, rbohD and rbohF mutants assessed at 3 days after syringe‐infiltration with Pst COR⁻ at 10⁶ cfu/ml (left panel) or after spray‐inoculation with Pst COR⁻ at 10⁸ cfu/ml (right panel). Values are the means ± SE (n = 6). (d) The rbohF mutant is partially defective in H₂O₂‐mediated stomatal closure. Stomatal apertures in WT Col‐0, rbohD and rbohF leaf discs exposed to Control or 100 μm H₂O₂ for 2 h. Data are means ± SE (n ≥ 60) from a representative experiment. (e) Dose response of Col‐0 WT stomata to H₂O₂ indicate that high H₂O₂ concentration does not close stomata. Stomatal apertures in WT Col‐0 leaf discs exposed to different concentrations of H₂O₂ (0, 10, 100 and 1000 μm) for 2 h. Data are means ± SE (n ≥ 60) from a representative experiment. Different letters indicate significant differences at P < 0.001 (A, B, D and E) and P < 0.05 (C) based on a Tukey's HSD test.

Figure 4

RBOHF influences apoplastic pH. (a) Apoplastic pH in Col‐0 WT, rbohD and rbohF mutant leaves. Apoplastic pH in untreated condition was determined by multiwell fluorimetry (sequential excitation at 440 ± 8 and 495 ± 8 nm; emission, 525 ± 20 nm) on Oregon green dextran‐infiltrated leaf discs from 5 week‐old plants (See Methods for details). Data are means ± SE (n ≥ 12) from a representative experiment. Different letters indicate significant differences at P < 0.001 based on a Tukey's HSD test. (b) Kinetics of flg22‐induced leaf apoplastic alkalinisation in Col‐0 WT, rbohD and rbohF mutants. Oregon green dextran‐infiltrated leaf discs were exposed at t = 0 min to control solution or 1 μm flg22, the apoplastic pH was measured over time by multiwell fluorimetry and expressed relative to the mean initial pH (pHi) before treatment (pH/pHi). Data are means ± SE (n ≥ 18) from 3 independent experiments. Significant differences at P < 0.05 between flg22‐treated Col‐0 and rbohF were found between 10 and 60 min based on two‐way ANOVA and uncorrected Fisher's LSD analyses for each time point. (c) Hypothetical model of the regulation of apoplastic pH by RBOHF during PTI activation. In normal unstressed condition, the efflux of electron (e⁻) produced by the NADPH oxidase activity is compensated by an efflux of proton (H⁺) probably generated by plasma membrane H + ATPases. Both the acidification of the apoplast and the production of hydroxyl radicals (OH^•) from superoxide (O₂ ⁻) through the Haber–Weiss and Fenton reactions contribute to cell expansion. The plant immune response may induce the inhibition of H⁺ATPases and the activation of RBOHF together with superoxide dismutases (SOD) or germin‐like proteins which dismutates O₂ ⁻ to H₂O₂, a reaction that consumes H⁺. The resulting alkalinisation of the apoplast induces a depolarisation of the plasma membrane ultimately leading to stomatal closure via the activation of ion channels.