Subcellular Dynamics in Plants Figure 10. BL-induced cell wall expansion is paralleled by changes in the BRI1-GFP fluorescence lifetime. (A) BL-induced cell wall expansion of BRI1-GFP expressing hypocotyl cells from Arabidopsis seedlings before (black circles) and 30 min after (green circles) hormone application. The fluorescence intensity was recorded over a 7.0 mm section as indicated by the white line in the confocal image inlet. (B) Fluorescence lifetimes of BRI1-GFP in the identical plasmalemma-cell wall section shown in A before (black squares), 10 (red squares), 20 (blue squares) and 30 min (green squares) after addition of 25 nM BL. The minima in the lifetime curves correspond to the maxima in the intensity profiles shown in A as indicated by the dashed lines. (C) Fluorescence lifetimes of BRI1-GFP in root tip cells 0 to 30 min after application of 25 nM BL. Lifetimes were obtained by integrating over all recorded pixels of the confocal image. (D) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) recorded over a 3.0 mm plasmalemma-cell wall section of a BRI1-GFP expressing hypocotyl cell before (black squares), 15 min (blue squares) and 30 min (green squares) after onset of mock treatment. (E) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) recorded over a 3.0 mm plasmalemma-cell wall section of an aquaporin-GFP expressing hypocotyl cell before (black squares) and 30 min (green squares) after application of 25 nM BL. For the calculation of the lifetime values and error bars see Material and Methods. The experiments in B and C were repeated three times using cells from three independent seedlings. Representative results are shown. The results of the additional BRI1-GFP lifetime measurements along plasmalemma-cell wall sections are presented in Figure S2. doi:10.1371/journal.pone.0005716.g010 response function (IRF) measured without the long pass filter and then fitted by a mono- or multi-exponential decay function. The cell physiological experiments (cell wall expansion, BRI1- GFP and aquaporin lifetime measurements) were repeated at least 3 times using independent seedling samples. Probe preparation and transmission electron microscopy (TEM) Seedlings were incubated in water or 10 nM brassinolide solution for 30 min at room temperature. Thereafter, root tips were high- pressure frozen and freeze-substituted in acetone containing 2.5% osmium tetroxide (60 h at 290uC, 8 h at 260uC and 8 h 235uC) before transfer for 60 min to 0uC [37]. Then, the root tips were washed five times with acetone followed by infiltration with 10% (4 h), 25% (12 h), 50% (12 h), 75% (12 h) and 100% (2 times for 15 h, each) epoxy resin. The resin was allowed to polymerise for 2 days at 60uC. Ultrathin sections of the root tip samples were stained with 3% uranyl acetate in ethanol and lead citrate and viewed in a LEO 906 transmission electron microscope [37]. 72 walls derived from two independent, mock-treated BRI1-GFP expressing seed- lings and 72 walls derived from two independent, BL-treated BRI1- GFP expressing seedlings were measured. Data analysis The standard deviations in the Gaussian fits of the intensity profiles, as represented by the error bars, reflect the accuracy of the fit and rest on the different signal-to-noise ratio of the measurements. The FWHM-based and TEM-based wall mea- surements of cells before/without and after treatment with BL were performed as indicated in the Tables S 2, S3, S4, S5, S6 and PLoS ONE | www.plosone.org 11 May 2009 | Volume 4 | Issue 5 | e5716