Subcellular Dynamics in Plants Figure 3. Measurement ranges for comparison between GFP-fluorescence and cell wall-derived autofluorescence. (A) Overlap of the fluorescence spectrum of purified GFP in water (green) and the autofluorescence spectrum (blue) of wildtype Arabidopsis hypocotyl cells. The broad band of the spectrum (500–640 nm, blue) originates from the cell wall, the sharp peak at 680 nm originates from chlorophyll. The grey hatched areas show the spectral ranges used for recording GFP-fluorescence (around 500 nm) and autofluorescence, respectively (around 600 nm). (B) Fluorescence decay trace recorded in the 500 nm region (left hatched bar in A) in a BRI1-GFP expressing hypocotyl cell, fitted by a mono-exponential function (black line), proves the main presence of GFP-fluorescence. (C) Fluorescence decay trace recorded in the 600 nm region (right hatched bar in A) in a BRI1-GFP expressing hypocotyl cell, fitted by a mono-exponential function (black line), proves the dominant presence of autofluorescence. IRF, instrument response function. doi:10.1371/journal.pone.0005716.g003 subcellular area was not yet studied. We therefore, recorded fluorescence intensity profiles over selected plasmalemmata-cell wall sections before and after treating the cells for 15 to 30 min with 10 nM BL. The intensity profiles enabled us to discriminate between the fluorescence signal of the proper plasmalemmata-cell wall section and membrane vesicles, which budded from the plasmalemmata during the incubation time. As shown in Fig. 4A– D and Fig. 5, the treatment of seedlings with BL induced an expansion of the BRI1-GFP signal in the plasmalemma-cell wall section of the root cells within 30 min. This response was also observed in hypocotyl cells, where BL induced an expansion of the BRI1-GFP signal of two neighboring cells, so that the plasma- lemmata became optically distinguishable (Fig. 4E and F). The degree of expansion did not only depend on the cell type but also on the position of the measured section along the cell wall of individual cells (Fig. 5). The Gaussian fitting of the GFP intensity profiles of more than 30 independent sections measured in several individual cells from 5 seedlings revealed that the fluorescence signals expanded around 34622% (n = 31, p = 0,0003) in response to BL treatment (see Table S2 for the single measurement values). The spatial separation of the plasmalemmata could have been caused by BL-induced plasmolysis or BL-induced cell wall expansion. Therefore, we determined the spatial behavior of the cell wall in response to BL treatment in BRI1-GFP-expressing seedlings. The measurement of fluorescence from different subcellular origins was possible as demonstrated before (Fig. 3). The cell wall autofluorescence expanded around 41624% (n = 29, p = 0,0145) after brassinolide treatment coincidentally with the separation of the plasmalemmata in the analysed cells (see Table S3 for the single measurement values and Fig. 6A as an example). PLoS ONE | www.plosone.org 4 May 2009 | Volume 4 | Issue 5 | e5716