Analysis of the leaf peeling and rapid fixation method for studies on stomatal responses.

<p>(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).</p

Visualization of stomatal apertures in intact leaves and epidermis peels.

<p>(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; λ_exc = 488 nm, λ_em = 505–545 nm (upper panel); λ_exc = 561 nm, λ_em = 600–640 nm (middle panel); bright field (lower panel). Bars, 50 μm.</p

Changes in stomatal apertures in <i>Arabidopsis</i> leaves.

<p>Stomata closure in response to 10 μM ABA and 100 μM H₂O₂ in Col-0, <i>ahk5-1</i> and AHK5 overexpressor lines (a) and Ws-4 and <i>ahk5-3</i> 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 <i>Arabidopsis</i> leaves. (c) Stomatal apertures in leaves treated with 10 μM ABA or 5 μM IAA for 2 h. <i>pre-st</i>.: pre-stained leaves, <i>post-st</i>.: 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).</p