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. 2020 Mar 24;10(1):5333.
doi: 10.1038/s41598-020-62239-w.

Hoechst-tagged Fluorescein Diacetate for the Fluorescence Imaging-based Assessment of Stomatal Dynamics in Arabidopsis Thaliana

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Free PMC article

Hoechst-tagged Fluorescein Diacetate for the Fluorescence Imaging-based Assessment of Stomatal Dynamics in Arabidopsis Thaliana

Yousuke Takaoka et al. Sci Rep. .
Free PMC article

Abstract

In plants, stomata regulate water loss through transpiration for plant growth and survival in response to various environmental stressors; and simple methods to assess stomatal dynamics are needed for physiological studies. Herein, we report a fluorescence-imaging-based method using fluorescein diacetate tagged with Hoechst 33342, a nuclear staining chemical probe (HoeAc2Fl) for the qualitative assessment of stomatal dynamics. In our method, the stomatal movement is inferred by simple monitoring of the fluorescence intensity in the nucleus of the stomata.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) Chemical structure of HoeAc2Fl which comprises Hoechst 33342 and fluorescein diacetate moieties. (b) Schematic illustration of selective staining of the nucleus in the closed stomata by HoeAc2Fl.
Figure 2
Figure 2
(a,b) Differential interference images (DIC) and fluorescent (tdTomato or Fluorescein) microscopic images of HoeAc2Fl-stained stomata of P35S ::H2B-tdTomato35S::H2B-tdTomato in the dark (a) or light (b) conditions; only the closed stomata were stained with HoeAc2Fl (the stomatal aperture was 2.15 µm), whereas opened stomata were not (the stomatal aperture was 4.70 µm) (see images in the fluorescein channel). The scale bars, 10 µm. (c) Relationship between stomatal apertures and fluorescent intensity of the nucleus of HoeAc2Fl-stained guard cells in the dark (black circle) or light condition (blue diamond). (d) Dotted plot of the fluorescence intensity of HoeAc2Fl-stained stomata in the dark (black circle) or light (blue diamond) conditions. Bars represent mean fluorescence intensity (n = 30). Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.01).
Figure 3
Figure 3
(a) Stomatal aperture of Col-0 during the staining process with HoeAc2Fl in the dark (0–90 min) and stomatal opening process in the light condition (total incubation time was 120-210 min, which is incubated for 30–120 min in the light condition). Error bars represent mean and SD (n = 25). (b) Dotted plot of the fluorescence intensity of staining process in the dark (0–90 min) and stomatal opening process in the light condition (total incubation time was 120–210 min, which is incubated for 30–120 min in the light condition) of HoeAc2Fl. Bars represent mean fluorescence intensity (n = 25). (c) Stomatal aperture of Col-0 during the staining process with HoeAc2Fl in the light (0–120 min) and stomatal closing process in the dark condition (total incubation time was 150–300 min, which is incubated for 30–180 min in the dark condition). Error bars represent mean and SD (n = 25). (d) Dotted plot of the fluorescence intensity of staining process in the light (0–120 min) and stomatal closing process in the dark condition (total incubation time was 150–300 min, which is incubated for 30–180 min in the dark condition) of HoeAc2Fl. Bars represent mean fluorescence intensity (n = 25). Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.01).
Figure 4
Figure 4
Fluorescent microscopic images of stomata of Col-0 stained by Hoechst 33342 (20 µg/mL, (a) or DAPI (20 µg/mL, (b) in the dark (top) or light (bottom) conditions. The scale bars, 10 µm.
Figure 5
Figure 5
(a) Fluorescent microscopic images of HoeAc2Fl-stained stomata of Col-0 treated with ABA (10 µM) in the light. The scale bars, 10 µm. (b) Stomatal aperture of Col-0 treated without or with ABA (10 µM). Error bars represent mean and SD (n = 25). (c) Relationship between stomatal apertures and fluorescence intensity of the nucleus of HoeAc2Fl-stained guard cells in the mock condition (black circle), in the absence (red cross) or the presence of ABA (blue diamond). (d) Dotted plot of the fluorescence intensity of HoeAc2Fl-stained stomata in the mock condition (black circle), in the absence (black cross) or the presence of ABA (black diamond). (eg) Fluorescent microscopic images of HoeAc2Fl-stained stomata of Col-0 treated with IAA (10 µM, e), COR (10 µM, f), or FC (10 µM, g) in the dark. The scale bars, 10 µm. (h) Stomatal aperture of Col-0 treated without or with various chemicals (IAA, COR, and FC, 10 µM). Error bars represent mean and SD (n = 25). Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.01). (i) Relationship between stomatal apertures and fluorescence intensity of the nucleus of HoeAc2Fl-stained guard cells in the mock condition (black circle) or treated with IAA (green square), COR (blue triangle), or FC (red cross). (j) Dotted plot of the fluorescence intensity of HoeAc2Fl-stained stomata in the mock condition (black circle) or treated with IAA (green square), COR (blue triangle), or FC (red cross). Bars represent mean fluorescence intensity (n = 25). Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.01).

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