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.

High lipid order of Arabidopsis cell‐plate membranes mediated by sterol and DYNAMIN‐RELATED PROTEIN1A function

“…Lipid order may be probed using fluorescent, lipid order-sensitive probes, both in artificial membranes in vitro (Jin et al, 2006;Owen et al, 2006) and in live cells in vivo (Owen et al, 2012). The fluorescent probe di-4-ANEPPDHQ has been validated for determining lipid order in extracted plant membranes and in live imaging of plant cells (Roche et al, 2008;Liu et al, 2009;Gerbeau-Pissot et al, 2013). Lipid order affects the fluorescence emission spectrum of di-4-ANEPPDHQ, with the spectral region between 500 and 580 nm representing the ordered phase of the membrane, and fluorescence between 620 and 750 nm representing the disordered phase (Owen et al, 2012).…”

Differential Effect of Plant Lipids on Membrane Organization

“…A further pixel-to-pixel analysis of red/green ratio (RGP, for red/green ratio of the pixel) was performed to map the order level of the membrane (Fig. 5A), as achieved previously on the cell membrane (67). When used on a classical animal lipid mixture, DOPC/sphingomyelin (SM)/cholesterol (4.5:4.5:1), this method allows observation of the partitioning of reddish and greenish regions indicative of the coexistence of large L o and L d areas (Fig.…”

The synthetic cationic lipid diC14 activates a sector of the Arabidopsis defence network requiring endogenous signalling components

“…Its insertion into the membrane may either disturb lipid compartmentalization, or generate curvature constraints, thus affecting mechanosensitive channels or proteins involved in defence signalling (Henry et al ., ). Cryptogein alters the PM of tobacco cells by modifying lateral compartmentalization and biophysical properties (fluidity), suggesting the generation of signalling platforms at the cell surface (Gerbeau‐Pissot et al ., ). In addition, cryptogein (Stanislas et al ., ), chitin (Fujiwara et al ., ) and flg22 (Keinath et al ., ) modify the protein composition of detergent‐resistant PM (DRM) fractions.…”