Modification of plasma membrane organization in tobacco cells elicited by cryptogein

Abstract

Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the "membrane raft" hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment.

Figure 1.

Characterization of the red/green ratio of a single-cell PM labeled with di-4-ANEPPDHQ by multispectral confocal microscopy. A, Observation of maximum surface area of di-4-ANEPPDHQ-labeled tobacco BY2 suspension cells (excitation at 488 nm; emission corresponds to the sum of fluorescence intensities acquired from all channels, ranging from 520 to 680 nm) with a color rendering of pixel fluorescence. B, Separate images for each wavelength corresponding to the emission fluorescence of a single channel captured by a 5-nm band pass (central value is reported). C, Representative emission spectra of di-4-ANEPPDHQ in tobacco cell PM exposed for 5 min at different temperatures (n = 10 independent cells for each condition). Depending on the device available, previous studies have used different band passes centered around 550 and 660 nm to quantify the blue shift of the dye on living material. For example, the horizontal, light gray bands correspond to 500 to 580 nm and 620 to 750 nm (Miguel et al., 2011), while the horizontal, dark gray bands correspond to 505 to 550 nm and a long pass higher than 650 nm (Liu et al., 2009). The colored, vertical band pass bars correspond to the values used in our experimental setup to calculate an accurate red/green ratio (545–565 nm in red and 635–655 nm in green).

Figure 2.

Increase of the global level of order at the PM surface of elicited tobacco cells. A, The time course of the RGM was followed after elicitation with 50 nm cryptogein (cry). B, The RGM was measured after 5 min treatment with 50 nm BSA, 100 nm lysozyme (lys), and 20 nm of flagellin (flg22) and compared to control cells (ctl). Data shown are mean values ± se of the mean (n = 14–66 cells). The asterisk indicates a significant difference (P value < 0.05).

Figure 3.

Influence of cryptogein treatment on the global fluidity of tobacco cell PM measured by FRAP. A, Normalized fluorescence recovery (excitation: 488 nm, emission: 510–700 nm) was plotted for control cells or cells elicited with 50 nm cryptogein (+cry) after 5 min of treatment. The fluorescence level is not normalized to the initial fluorescence, and only the mobile fraction is represented (final percentage: 100%). The plotted control and cryptogein lines represent results of fit to F(t) = (F₀ + F∞ × [t/t_1/2])/(1 + [t/t_1/2]), where F₀ is the immediate postbleach intensity, F∞ is the asymptote of fluorescence recovery, and t_1/2 is the time required to recover 50% of the asymptote fluorescence (error bars indicate ± se of the mean). B, Influence of cryptogein elicitation treatment (cry) on half-maximal recovery. The asterisk denotes a statistically significant difference (P value < 0.01), and the data shown are mean values ± se of the mean. For control cells, n = 58 (5 min) and 40 (15 min); for treated cells, n = 89 (5 min) and 52 (15 min). C, Influence of the treatments 50 nm BSA (n = 25), 100 nn lysozyme (lys; n = 36), and 20 nm flagellin (flg22; n = 28) on half-maximal recovery, compared with a control (ctl; n = 48). The asterisk denotes a statistically significant difference (P value < 0.01), and the data shown are mean values ± se of the mean.

Figure 4.

Analysis of different levels of order within the PM of tobacco cells. A, Comparison of PM order level, using RGM (the red/green ratio calculated from the global emission spectrum exhibited by the entirety of imaged cell membrane areas) or mean RGR (the average of the ratios of individual ROI [288 nm × 288 nm] of the cell surface area; mean ± se of the mean, n = 49 cells). B, Comparison of RGR distribution from a single cell (all ROIs) with RGR distributions corresponding to the 10% of ROIs exhibiting highest fluorescence intensity (over the 90th percentile value); ROIs exhibiting fluorescence intensity above 80% of the maximum fluorescence intensity measured (>80%); and randomly selected ROIs from the same range as the greater than 80% sample (Random).

Figure 5.

Heterogeneity of the order level of tobacco cell PM is sterol sensitive. A, Distribution of red/green ratio values of individual ROIs (288 nm × 288 nm) of the PM and influence of sterol depletion (+MβCD). The x axis corresponds to the class of RGR values, and only the maximal value of each class is reported on the graph. The y axis corresponds to the ROI percentage of each class. B, Influence of sterol depletion (+MβCD) on RGM. C, Representativeness of low RGR values according to PM sterol content. ROIs exhibiting an RGR below the threshold values indicated on the x axis were counted, and their percentage was reported along the y axis for control (white) and treated (black) cells. For both B and C, asterisks indicate a significant difference (P value < 0.01), and data are means ± se of the mean (n = 49 [control] and 11 [treated cells]).

Figure 6.

Increase of the proportion of ROIs with low RGR at the PM surface of cryptogein-elicited tobacco cells. Ratiometric images of tobacco BY2 suspension cells that were either elicited with cryptogein (bottom) or not (top) are displayed in A and B. A, A pseudocolor-coded representation of total cell surface area, according to the accompanying RGR scale. B, Zoom of an area extracted from the plant membrane surface (ROI size: 288 nm × 288 nm). C, Comparison of RGR distribution between cryptogein-treated (black triangles) and control cells (white squares). The x axis represents the class of RGRs values; only the maximal value of each class is reported on the graph. The y axis represents the percentage of each class of ROI values. Data are means ± se of the mean (n = 49 cells for both conditions).