Subcellular Dynamics in Plants Figure 4. BL-induced expansion of the GFP fluorescence in BRI1-GFP expressing root tips and hypocotyl cells. (A–C) Fluorescence intensity images and the corresponding profiles (Gaussian fits including FWHM values) of root tip cells recorded over the subcellular section indicated by the white line (representing 5 mm) shown in the confocal image inlets before (0 min) A, 15 min, B, and 30 min, C, after addition of 10 nM BL. (D) Combined Gaussian fits of the intensity profiles shown in A–C. (E–F) Fluorescence intensity profiles recorded over a 5 mm section of the plasmalemmata-cell wall area of two neighbouring hypocotyl cells before, E, and 30 min after application of 10 nM BL, F. For the determination of the FWHM error see Material and Methods. For the statistical analysis of the BRI1-GFP fluorescence measurements see Table S2. doi:10.1371/journal.pone.0005716.g004 A BL-induced expansion of cell wall by 49629% (n = 15, p = 0,0131) was also observed when the BRI1-GFP expressing seedlings were stained with the cell wall-staining dye Calcofluor (Fig. 6B, see Table S4 for the single measurement values). We also carried out a TEM analysis of control and BL-treated (30 min) root cells. Root tips from two seedlings of each were frozen under high pressure, freeze-substituted in osmium tetroxide containing acetone, embedded in epoxy resin, cut and subjected to TEM. Using the TEM images, the width of 72 periclinal cell walls of each was measured, and the mean width calculated. The measurements resulted in a wall width of 62,5610,8 nm for the control and 64,9610,1 nm for BL-treated root tip cells (Fig. 7; see Table S 5 for the single measurement values). Thus, a statistically significant difference with high confidence between BL-treated and mock-treated seedlings (p = 0,0845) could not be observed by using TEM. We realized, however, that the variation of the wall width between individual cells and within an individual cell was high and ranged from 47 to 88 nm in the mock-treated and from 49 to 94 nm in the BL-treated root tips (Table S 5; see Discussion for the set of difficulties with respect to the TEM measurements). In addition, our data showed that the cellular structures including the plasmalem- mata and cell walls were normal in BL-treated as well as in untreated cells (Figs. 2 and 7). Furthermore, there was no detachment of the plasmalemmata from the cell wall in response to BL treatment (Fig. 7). The latter observation excludes a possible plasmolytic cause for the BL-induced expansion in the BRI1-GFP signal. To determine the specificity of the response, we carried out identical experiments on seedlings, expressing plasmalemma- located aquaporin-GFP [28] and wildtype seedlings. Our experiments revealed a slight BL-induced separation of adjacent GFP signals by 1068% (see Table S6 for statistics and single measurement values; Fig. 8A–F) in aquaporin-GFP expressing seedlings and a slight expansion of the cell wall autofluorescence by 18616% in wild type seedlings (see Table S7 for statistics and single measurement values) without general statistical significance. We obtained similar results, when Calcofluor-stained cell walls or the autofluorescence of the cell walls in aquaporin-GFP-expressing cells were measured (Fig. 8G–H; data not shown). PLoS ONE | www.plosone.org 5 May 2009 | Volume 4 | Issue 5 | e5716