Stomata Prioritize Their Responses to Multiple Biotic and Abiotic Signal Inputs

<div><p>Stomata are microscopic pores in leaf epidermis that regulate gas exchange between plants and the environment. Being natural openings on the leaf surface, stomata also serve as ports for the invasion of foliar pathogenic bacteria. Each stomatal pore is enclosed by a pair of guard cells that are able to sense a wide spectrum of biotic and abiotic stresses and respond by precisely adjusting the pore width. However, it is not clear whether stomatal responses to simultaneously imposed biotic and abiotic signals are mutually dependent on each other. Here we show that a genetically engineered <i>Escherichia coli</i> strain DH5α could trigger stomatal closure in <i>Vicia faba</i>, an innate immune response that might depend on NADPH oxidase-mediated ROS burst. DH5α-induced stomatal closure could be abolished or disguised under certain environmental conditions like low [CO₂], darkness, and drought, etc. Foliar spraying of high concentrations of ABA could reduce stomatal aperture in high humidity-treated faba bean plants. Consistently, the aggressive multiplication of DH5α bacteria in <i>Vicia faba</i> leaves under high humidity could be alleviated by exogenous application of ABA. Our data suggest that a successful colonization of bacteria on the leaf surface is correlated with stomatal aperture regulation by a specific set of environmental factors.</p></div

DH5α-triggered stomatal closure is disguised by darkness or drought treatment.

<p>A and B, Stomatal aperture and conductance in <i>V. faba</i> leaves dip-inoculated with mock or DH5α at 10⁸ CFU/ml under light/dark transition; C and D, Stomatal aperture and conductance in <i>V. faba</i> leaves dip-inoculated with mock or DH5α at 10⁸ CFU/ml under different field water content (FWC). Data from the epidermal bioassay are means of 120 stomatal aperture measurements from three replicates ±SEM. Data from the stomatal conductance experiment are means of measurements from 8–12 leaves (n = 4). Asterisks denote significant differences as analyzed by two-tailed <i>t</i>-test (***, <i>P</i><0.001; *, <i>P</i><0.05; ns, no statistical difference).</p

DH5α-induced stomatal closure involves ROS accumulation in guard cells.

<p>A, Stomatal aperture in <i>V. faba</i> epidermal peels incubated with different treatments. Data are means of 120 stomatal aperture measurements from three replicates ±SEM; B, ROS accumulation in intact guard cells detected by H₂DCF-DA fluorescence. The microscopic images represent fluorescent and DIC images of peels treated with mock (upper left and right), fluorescent image of peels treated with DH5α at 10⁸ CFU/ml (lower left), and fluorescent image of peels treated with 1 mM LPS (lower right). Bars  =  50 µm; C, Quantitation of generated ROS in <i>Vicia faba</i> guard cells as shown in B. Asterisks denote significant differences as analyzed by two-tailed <i>t</i>-test (***, <i>P</i><0.001; ns, no statistical difference).</p

DH5α-elicited stomatal closure is abolished by low [CO₂] or high RH treatment.

<p>A and B, Stomatal aperture and conductance in <i>V. faba</i> leaves dip-inoculated with mock or DH5α at 10⁸ CFU/ml under ambient or low CO₂ concentrations; C and D, Stomatal aperture and conductance in <i>V. faba</i> leaves dip-inoculated with mock or DH5α at 10⁸ CFU/ml under ambient and high RH conditions. Data from the epidermal bioassay are means of 120 stomatal aperture measurements from three replicates ±SEM. Data from the stomatal conductance experiment are means of measurements from 8–12 leaves (n = 4). Asterisks denote significant differences as analyzed by two-tailed t-test (***, P<0.001; **, P<0.01; *, P<0.05; ns, no statistical difference).</p

Exogenous ABA can reduce stomatal aperture and inhibit foliar bacterial growth under high RH.

<p>A, Stomatal aperture in <i>V. faba</i> leaves dip-inoculated with mock or DH5α (10⁸ CFU/ml) supplemented with the indicated concentrations of ABA under ambient and high RH conditions. Results represent means of three replicates ±SEM, (n = 120 stomata). “+” and “−” represent presence or absence of DH5α cells in the inoculum. Asterisks denote significant differences as analyzed by two-tailed <i>t</i>-test (**, <i>P</i><0.01; ns, no statistical difference); B, Bacterial population in <i>V. faba</i> leaves at day 3 after dip inoculation with DH5α. “+” and “−” represent presence or absence of ABA (20 µM) in the inoculum.</p

<i>E. coli</i> DH5α can trigger stomatal closure in <i>V. faba</i>.

<p>A, Stomatal aperture in <i>V. faba</i> epidermal peels incubated with DH5α at the indicated concentrations; B, Stomatal aperture in <i>V. faba</i> epidermal peels incubated with mock or DH5α at 10⁸ CFU/ml. Results represent means of three replicates ±SEM, (n = 120 stomata).</p

Pathogenicity of DC3118 in <i>Arabidopsis</i> can be modulated by extrinsic factors.

<p>A, Stomatal aperture in Col-0 leaves surface-inoculated with mock or DC3118 at 10⁸ CFU/ml under ambient or high RH; B, Stomatal aperture in Col-0 leaves surface-inoculated with different treatments under high RH. Data in A and B represent means of 120 stomatal aperture measurements from three replicates ±SEM. Asterisks denote significant differences as analyzed by two-tailed <i>t</i>-test (***, <i>P</i><0.001; ns, no statistical difference); C, Progression of disease symptom in Col-0 plants with the following treatments: (I) RH = 60%, mock; (II) RH≥90%, mock; (III) RH = 60%, DC3118 (10⁸ CFU/ml); (IV) RH≥90%, DC3118 (10⁸ CFU/ml); (V) RH≥90%, DC3118 (10⁸ CFU/ml) + ABA (20 µM).</p

Data_Sheet_1_Effects of 5-azaC on Iridoid Glycoside Accumulation and DNA Methylation in Rehmannia glutinosa.docx

Iridoid glycoside is the important secondary metabolite and the main active component in Rehmannia glutinosa. However, the mechanisms that underlie the regulation of iridoid glycoside biosynthesis remain poorly understood in R. glutinosa. Herein, the analysis of RNA-seq data revealed that 3,394 unigenes related to the biosynthesis of secondary metabolites were identified in R. glutinosa. A total of 357 unigenes were involved in iridoid glycoside synthesis, in which the highly conservative genes, such as DXS, DXR, GPPS, G10H, and 10HGO, in organisms were overexpressed. The analysis of the above genes confirmed that the co-occurrence ratio of DXS, DXR, and GPPS was high in plants. Further, our results showed that under normal and 5-azacytidine (5-azaC) treatment, the expression levels of DXS, DXR, GPPS, G10H, and 10HGO were consistent with the iridoid glycoside accumulation in R. glutinosa, in which the application of the different concentrations of 5-azaC, especially 50 μM 5-azaC, could significantly upregulate the expression of five genes above and iridoid glycoside content. In addition, the changes in the spatiotemporal specificity of degree and levels of DNA methylation were observed in R. glutinosa, in which the hemi-methylation was the main reason for the change in DNA methylation levels. Similar to the changes in 5-methyl cytosine (5mC) content, the DNA demethylation could be induced by 5-azaC and responded in a dose-dependent manner to 15, 50, and 100 μM 5-azaC. Taken together, the expression of iridoid glycoside synthesis gene was upregulated by the demethylation in R. glutinosa, followed by triggering the iridoid glycoside accumulation. These findings not only identify the key genes of iridoid glycoside synthesis from R. glutinosa, but also expand our current knowledge of the function of methylation in iridoid glycoside accumulation.</p

Data_Sheet_2_Effects of 5-azaC on Iridoid Glycoside Accumulation and DNA Methylation in Rehmannia glutinosa.xlsx

Iridoid glycoside is the important secondary metabolite and the main active component in Rehmannia glutinosa. However, the mechanisms that underlie the regulation of iridoid glycoside biosynthesis remain poorly understood in R. glutinosa. Herein, the analysis of RNA-seq data revealed that 3,394 unigenes related to the biosynthesis of secondary metabolites were identified in R. glutinosa. A total of 357 unigenes were involved in iridoid glycoside synthesis, in which the highly conservative genes, such as DXS, DXR, GPPS, G10H, and 10HGO, in organisms were overexpressed. The analysis of the above genes confirmed that the co-occurrence ratio of DXS, DXR, and GPPS was high in plants. Further, our results showed that under normal and 5-azacytidine (5-azaC) treatment, the expression levels of DXS, DXR, GPPS, G10H, and 10HGO were consistent with the iridoid glycoside accumulation in R. glutinosa, in which the application of the different concentrations of 5-azaC, especially 50 μM 5-azaC, could significantly upregulate the expression of five genes above and iridoid glycoside content. In addition, the changes in the spatiotemporal specificity of degree and levels of DNA methylation were observed in R. glutinosa, in which the hemi-methylation was the main reason for the change in DNA methylation levels. Similar to the changes in 5-methyl cytosine (5mC) content, the DNA demethylation could be induced by 5-azaC and responded in a dose-dependent manner to 15, 50, and 100 μM 5-azaC. Taken together, the expression of iridoid glycoside synthesis gene was upregulated by the demethylation in R. glutinosa, followed by triggering the iridoid glycoside accumulation. These findings not only identify the key genes of iridoid glycoside synthesis from R. glutinosa, but also expand our current knowledge of the function of methylation in iridoid glycoside accumulation.</p