Subcellular Dynamics in Plants Figure 5. BL-induced expansion of the GFP fluorescence signal in BRI1-GFP expressing root tip cells. (A) Confocal image of root tip tissue before (left) and 30 min after addition of 10 nM BL (right). Fluorescence intensity curves were recorded over the plasmalemmata-cell wall sections indicated by the white, alphabetically numbered lines and the FWHM values of their Gaussian fits calculated before and 30 min after addition of BL. (B–J) FWHM values of the plasmalemmata-cell wall sections indicated in A before (0 min) and 30 min after addition of 10 nM BL. For the determination of the FWHM error in B to J see Material and Methods. For the statistical analysis of the BRI1-GFP fluorescence measurements see Table S2. doi:10.1371/journal.pone.0005716.g005 Monitoring the environment of plasmalemma-bound GFP fusion proteins in vivo The lifetime of GFP fluorescence provides information about the physical and chemical environment of the fluorophore and, thus, the GFP fusion protein [29–30]. In addition to fluorescence intensity, we recorded GFP fluorescence lifetime decay traces across plasmalemmata-cell wall sections in hypocotyl cells of aquaporin-GFP and BRI1-GFP expressing seedlings at up to 200 nm intervals. The measurements were based on a different but sufficient number of fluorescence counts (Table S1), which is reflected in the error calculation [31]. We observed significant differences in GFP fluorescence lifetime across plasmalemma-cell wall sections and regularly also between adjacent cells for both aquaporin-GFP and BRI1-GFP (Fig. 9A and B). A detailed subcellular analysis revealed that the most significant differences in BRI1-GFP lifetime existed within the cell between the cytoplasm, plasmalemma and cell wall (Fig. 9C). To determine whether BL also induces changes in the environment of BRI1-GFP, we analysed the fluorescence lifetime of the receptor fusion. After the addition of BL, we again observed cell wall expansion (Fig. 10A). Furthermore, BRI1-GFP fluores- cence lifetime at the plasmalemmata strongly decreased within 20 min of BL addition and then remained constant (Fig. 10B, C; Figure S2). In contrast, fluorescence lifetime in the subcellular areas adjacent to the plasmalemma region remained largely unchanged (Fig. 10B). By the spatial extension of our measure- ments, BL-induced changes in BRI1-GFP lifetime were not only observed in sections over the plasmalemma-wall areas but also over larger cell areas (Fig. 10C). Significant changes in GFP fluorescence lifetime were never measured after mock-treatment of the BRI1-GFP line or in the BL-treated aquaporin-GFP expressing cells (Fig. 10D and E; data not shown). BR-induced responses are cell physiological processes Recent studies suggested that the endosomal pool of BRI1 is critical for the signaling and regulation of BL-responsive genes in Arabidopsis as shown by treatment with Brefeldin A (BFA) [22–23]. BFA inhibits the function of ARF-GTPases by interacting with their associated GEFs and, thus, has strong effects on the integrity of subcellular compartments and the endosomal vesicle pool by inhibiting intracellular trafficking pathways [32–33]. We, there- fore, addressed the question whether BFA treatment also interferes with BL-induced cell wall expansion and BRI-GFP fluorescence lifetime. In the presence of the inhibitor BFA compartments appeared and both processes were strongly inhibited, suggesting that an intact intracellular trafficking system is required for their execution (Fig. 11 and Figure S3). PLoS ONE | www.plosone.org 6 May 2009 | Volume 4 | Issue 5 | e5716