Figure 1.
Differentiation of autofluorescence and the GFP signal in BRI1-GFP-expressing and wildtype Arabidopsis cells.
(A–B) Wavelength-resolved fluorescence spectra from plasmalemma-cell wall sections of BRI1-GFP-expressing cells (green) and wildtype cells (black). The spectra were recorded in root, A, or cotyledon cells, B. The areas (50×50 µm) used for recording are shown in the inlets. (C) In vivo decay trace of fluorescence lifetime from BRI1-GFP expressing hypocotyl cells fitted by a mono-exponential function (black line). The lifetime decay was measured at the peak of fluorescence intensity at 500 to 512 nm. (D) In vivo decay trace of autofluorescence lifetime in the plasmalemma-cell wall area of wildtype hypocotyl cells fitted by a mono-exponential function (black line). (E) In vivo decay trace of autofluorescence lifetime in the plasmalemma-cell wall area of wildtype hypocotyl cells fitted by a multi-exponential function (black line). The lifetime decay was measured at around 500 to 512 nm. 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.
Figure 2.
Dimensional appearance of periclinal and anticlinal cell walls in confocal and TEM images.
(A) Fluorescence intensity curve recorded over a plasmalemmata-wall section of the periclinal wall of BRI1-GFP expressing Arabidopsis root cells. The full width at half maximum value (FWHM) of the Gaussian fitting revealed an apparent wall thickness of 425±30 nm. (B) Fluorescence intensity curve recorded over a plasmalemmata-wall section of the anticlinal wall of the identical root cell described in A. The FWHM of the Gaussian fitting revealed an apparent wall thickness of 773±33 nm. (C) Ultrathin TEM image of a tissue section cutting three different root cells. Two anticlinal and one periclinal cell walls (CW) are shown. CO, cortical cell; EP, epidermal cell; ER, endoplasmatic reticulum; EB, ER body; M, mitochondrium; V, vacuole. The arrow heads point to Golgi-derived vesicles. The bar represents 500 nm.
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.
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 µm) 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 µm 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.
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.
Figure 6.
BL-induced expansion of the cell wall in BRI1-GFP expressing hypocotyl cells.
(A) FWHM values of the anticlinal wall autofluorescence from hypocotyl cells recorded over the subcellular section indicated by the white line (10 µm) shown in the confocal image inlet at 0 min and 30 min after addition of 10 nM BL. (B) FWHM values of Calcofluor-stained anticlinal wall from hypocotyl cells recorded over the subcellular section indicated by the white line (11 µm) shown in the confocal image inlet at 0, 15 and 30 min after addition of 10 nM BL. For the determination of the FWHM error in A and B see Material and Methods. For the statistical analysis of the cell wall autofluorescence and Calcofluor fluorescence measurements see Tables S3 and S4.
Figure 7.
TEM images of ultrathin sections from different root cells of BRI1-GFP expressing seedlings.
(A) Ultrathin section of a root cell from a seedling high-pressure frozen 30 min after the addition of 10 nM BL. (B) Ultrathin section of an independent root cell from a mock-treated, high-pressure frozen seedling. The cell walls of epidermal cells (upper cells) facing cortex cells (lower cell) are shown. PM, plasmalemma; CW, cell wall; PD, plasmadesmon. The bar represents 100 nm. For the statistical analysis of the cell wall measurements see Table S5.
Figure 8.
BL-induced changes in the cell wall width of aquaporin-GFP expressing Arabidopsis 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. (B–F) FWHM values of the plasmalemmata-cell wall sections indicated in A before (0 min) and 30 min after addition of 10 nM BL. (G) FWHM values of Calcofluor-stained anticlinal wall from hypocotyl cells recorded over the subcellular section indicated by the white line (8 µm) shown in the confocal image inlet at 0, 15 and 30 min after addition of 10 nM brassinolide. (H) FWHM values of the anticlinal wall autofluorescence from hypocotyl cells recorded over the subcellular section indicated by the white line (5 µm) shown in the confocal image inlet at 0 min and 30 min after addition of 10 nM BL. For the determination of the FWHM error in B to H see Material and Methods. For the statistical analysis of the aquaporin-GFP fluorescence measurements see Table S6.
Figure 9.
ocFLM of BRI1-GFP and aquaporin-GFP reveals gradients in their subcellular environment.
(A) GFP fluorescence lifetimes (filled squares) and the corresponding intensity profiles (open circles) recorded over 5.0 µm plasmalemma-cell wall sections of two different hypocotyl cells from two independent BRI1-GFP expressing Arabidopsis seedlings. (B) GFP fluorescence lifetimes (filled squares) and the corresponding intensity profiles (open circles) recorded over 4.0 µm plasmalemma-cell wall sections of two different hypocotyl cells from two independent aquaporin-GFP expressing Arabidopsis seedlings. (C) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) over a 6.0 µm plasmalemma-cell wall area of a hypocotyl cell from a BRI1-GFP expressing Arabidopsis seedling. The white line in the confocal image inlet shows the recorded section. For the calculation of the fluorescence lifetime values and error bars see Material and Methods. Additional BRI1-GFP and aquaporin-GFP fluorescence lifetime measurements are presented in Fig. 10D and E.
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 µm 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 µm 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 µm 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.
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 µm plasmalemmata-cell wall section of hypocotyl cells from BRI1-GFP expressing Arabidopsis seedlings in the presence of 50 µM 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 µM 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 µM BFA. The BFA compartments are indicated by white arrows. The white bars represent 6 µm (left) and 10 µm (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.