Subcellular Dynamics in Plants Figure 11. BL-induced wall expansion and changes in BRI1-GFP fluorescence lifetime require a functional intracellular trafficking system. (A) FWHM values of GFP intensity profiles recorded over a 4.0 mm plasmalemmata-cell wall section of hypocotyl cells from BRI1-GFP expressing Arabidopsis seedlings in the presence of 50 mM BFA before (0 min) and 30 min after application of 10 nM BL. For the determination of the FWHM error see Material and Methods. (B) Fluorescence lifetimes of BRI1-GFP in the identical plasmalemma-cell wall section shown in A in the presence of 50 mM BFA before (black squares) and 30 min (green squares) after addition of 25 nM BL. For the calculation of the lifetime values and error see Material and Methods. (C) Confocal images of hypocotyl cells treated with 50 mM BFA. The BFA compartments are indicated by white arrows. The white bars represent 6 mm (left) and 10 mm (right). The experiments in A and B were repeated four times using cells from four independent seedlings. One representative result is presented. The results of additional measurements are shown in Figure S3. doi:10.1371/journal.pone.0005716.g011 S7. For statistical evaluation, the data were analysed by one-sided t-test. The number of independent measurements (n), the p-values and the number of independent seedlings are given in the legends of the Tables S2, S3, S4, S5, S6 and S7 and in Results. The lifetime values derived from mono- or multi-exponential decay fittings. The error bars were calculated according to [31] taking into account the number of photons and the time resolution of the measurements. The BRI1-GFP and aquaporin-GFP fluorescence lifetime measurements were repeated at least three times using individual cells from at least three independent seedlings and representative results are shown. The results of the additional experiments are presented in the Figures S2 and S3. Supporting Information Figure S1 Reference fluorescence spectrum and lifetime decay rate of purified GFP. (A) Fluorescence spectrum of GFP at a concentration of 1025 M after pulse excitation with light of 473 nm. The spectrum shows a peak at 510 nm and a shoulder at 540 nm. (B) Fluorescence decay rate of GFP at a concentration of 1027 M in 20 mM TRIS (pH = 6.8) after pulse excitation with light of 473 nm. The decay shows a mono-exponential function. The residuals indicate the deviation between the measured and the model decay function. In a good fit the residuals are distributed symmetrically around 0. IRF, instrument response function. Found at: doi:10.1371/journal.pone.0005716.s001 (0.07 MB PDF) Figure S2 BL induces changes in the BRI1-GFP fluorescence lifetime in plant cells. (A–B) Fluorescence lifetimes of BRI1-GFP in 4.0 mm plasmalemma-cell wall sections of two hypocotyl cells from two independent seedlings (A, B) before (black squares) and 10 (red squares), 20 (blue squares) and 30 min (green squares) after addition of 25 nM BL. Found at: doi:10.1371/journal.pone.0005716.s002 (0.08 MB PDF) Figure S3 BL-induced cell wall expansion and change in BRI1- GFP fluorescence lifetime require a functional intracellular trafficking system. (A–C) FWHM values of GFP intensity profiles (left) and fluorescence lifetime decays (right) recorded over plasmalemmata-cell wall sections of three hypocotyl cells (A, B, C) from three independent, BRI1-GFP expressing Arabidopsis seedlings in the presence of 50 mM BFA before (black) and 30 min (green) after application of 25 nm BL. Found at: doi:10.1371/journal.pone.0005716.s003 (0.15 MB PDF) Table S1 Fitting parameters of lifetime decay traces. Found at: doi:10.1371/journal.pone.0005716.s004 (0.17 XLS) MB Table S2 FWHM changes in the widening of the plasmalemma- bound GFP fluorescence signal in BRI1-GFP expressing hypocotyl and root cells before (0 min) and 30 min after application of 10 nM BL (30 min.). PLoS ONE | www.plosone.org 12 May 2009 | Volume 4 | Issue 5 | e5716