Fig 1.
Experimental setup for stomatal aperture measurements.
(a) Schematic representation of the workflow; (b) epifluorescent microscopic picture of the Arabidopsis leaf stained with rhodamine 6G; (c) the same picture as in (b) after application of the option “sharpen” in ImageJ. Bars, 50 μm.
Fig 2.
Changes in stomatal apertures in Arabidopsis leaves.
Stomata closure in response to 10 μM ABA and 100 μM H2O2 in Col-0, ahk5-1 and AHK5 overexpressor lines (a) and Ws-4 and ahk5-3 mutant line (b). Three fully expanded leaves at a similar developmental stage (one leaf per plant) per treatment were analyzed for each line. A single asterisk indicates a significant difference to corresponding mock-treated leaves (*, 0.01 < p ≤ 0.05), two asterisks depict a very significant difference (**, 0.001 < p ≤ 0.01), and three asterisks indicate an extremely significant difference to corresponding mock-treated leaves (***, p ≤ 0.001). (c,d) Comparison of stomatal movements in rhodamine 6G pre-stained (before treatment with hormones) and post-stained Arabidopsis leaves. (c) Stomatal apertures in leaves treated with 10 μM ABA or 5 μM IAA for 2 h. pre-st.: pre-stained leaves, post-st.: post-stained leaves. (d) Stomatal apertures in leaves treated with 10 μM ABA for 30 min and 1 h. A total of six leaves (c) and nine leaves (d) at a similar developmental stage (one leaf per plant) were used for the analyses. A very significant difference between leaves before treatment (0 h) and after treatment is indicated by two asterisks (**, 0.001 < p ≤ 0.01), an extremely significant difference is indicated by three asterisks (***, p ≤ 0.001).
Fig 3.
Visualization of stomatal apertures in intact leaves and epidermis peels.
(a,b) Epifluorescent images of intact leaves mounted in water (upper panel) and in 30% glycerol (lower panel). (a) Staining with 1 μM rhodamine 6G; (b) staining with 10 μM rhodamine 6G. (c) Photograph of leaf epidermis peels. (d) Confocal image of cells in the peeled epidermis; λexc = 488 nm, λem = 505–545 nm (upper panel); λexc = 561 nm, λem = 600–640 nm (middle panel); bright field (lower panel). Bars, 50 μm.
Fig 4.
Analysis of the leaf peeling and rapid fixation method for studies on stomatal responses.
(a) Stomatal closure in response to 2 h ABA treatment in intact leaves and epidermis peels. Dark-blue rectangles, mock; light-blue rectangles, 10 μM ABA. (b) Kinetics of stomatal closure in response to ABA in intact leaves and epidermis peels. A total of twelve leaves (one leaf per plant at a comparable developmental stage) have been used in each analysis. Two asterisks indicate a very significant difference to corresponding mock-treated cells (**, 0.001 < p ≤ 0.01), while three asterisks depict extremely significant difference to corresponding mock-treated cells (***, p ≤ 0.001). (c) Confocal images of cells in the peeled epidermis after quick fixation in 4% formaldehyde captured in red and bright field. Bar, 50 μm. (d,e) Comparison of stomatal apertures in fixed and non-fixed epidermis peels. (d) After 2 h pre-incubation in light the peels were analyzed under microscope, transferred into darkness for 30 min. and again analyzed. (e) The peels were pre-incubated in darkness, analyzed under microscope, transferred to light for 30 min. and analyzed again. Twelve leaves at a comparable developmental stage were used for peel preparations and subsequent analyses. Two asterisks indicate a very significant difference (**, 0.001 < p ≤ 0.01), while three asterisks depict extremely significant difference between the results of two subsequent measurements (***, p ≤ 0.001).