Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence

Abstract

Although plants are exposed to a great number of pathogens, they usually defend themselves by triggering mechanisms able to limit disease development. Alongside signalling events common to most such incompatible interactions, modifications of plasma membrane (PM) physical properties could be new players in the cell transduction cascade. Different pairs of elicitors (cryptogein, oligogalacturonides, and flagellin) and plant cells (tobacco and Arabidopsis) were used to address the issue of possible modifications of plant PM biophysical properties induced by elicitors and their links to other events of the defence signalling cascade. We observed an increase of PM order whatever the elicitor/plant cell pair used, provided that a signalling cascade was induced. Such membrane modification is dependent on the NADPH oxidase-mediated reactive oxygen species production. Moreover, cryptogein, which is the sole elicitor able to trap sterols, is also the only one able to trigger an increase in PM fluidity. The use of cryptogein variants with altered sterol-binding properties confirms the strong correlation between sterol removal from the PM and PM fluidity enhancement. These results propose PM dynamics as a player in early signalling processes triggered by elicitors of plant defence.

Keywords: Cryptogein mutants; elicitors; fluidity; membrane order; plant defence; plasma membrane; reactive oxygen species; signalling..

Fig. 1.

Increase of the PM order is associated with signalling triggered by elicitors of defence. Tobacco (A–C) or Arabidopsis (D–F) cells were exposed to cryptogein (Cry, 50nM), flagellin (Flg22, 20nM), oligogalacturonides (OG, 50ng ml⁻¹), or lysozyme (Lys, 100nM) and compared with control cells (Ctl, without treatment). (A, D) After a 5min treatment, cells were labelled with 3 µM di-4-ANEPPDHQ and emission spectra [control (dark line) and elicited (dotted line) cells] were recorded to quantify membrane order modifications using the red/green ratio (RGM, with RGM=I660/I550). (B, E) ROS production was assessed by chemiluminescence and followed during the first 60min after elicitation treatment. The sum of ROS produced during the kinetics was reported relative to the cryptogein-induced (B) or flagellin-induced (E) values (Arbitrary Units). (C, F) Extracellular alkalinization was reported as pH variation after 1h of treatment. Mean values ±SEM (n>3 independent experiments). Asterisks highlighted a significant difference compared with the control (P-value<0.05).

Fig. 2.

Increase of the representativeness of PM ordered domains by flg22 in Arabidopsis thaliana cells. Arabidopsis cells were observed after elicitation treatment with 20nM flg22 (bottom) or in control conditions (top). (A) Differential interference contrast (DIC) microscopy images. (B) Fluorescence (Fluo.) of plasma membrane of di-4-ANEPPDHQ-labelled Arabidopsis cells (excitation at 488nm; emission corresponds to the sum of fluorescence intensities acquired from channels ranging from 520nm to 680nm) with a grey scale rendering of fluorescence intensity. (C) Ratiometric images of di-4-ANEPPDHQ- (3 µM) labelled Arabidopsis cells were pseudocolour-coded, according to the accompanying RGP value scale showing the membrane order. A zoom of an area extracted from the PM surface is displayed. Scale bar=10 µm. (D) Each pixel constituting the analysed PM surface was associated with its own level of order (RGP) and a comparison of the distribution of the membrane order between flagellin-treated (Flg22, grey squares) and control cells (Ctl, black squares) is given. The x-axis represents the class of order level values. The y-axis represents the percentage of each class of pixel values. Data are means ±SE of the mean (n=206 cells, from four independent experiments).

Fig. 3.

Elicitor-induced increase of PM order depends on ROS production. (A) Effect of signalling inhibitors (staurosporin, Stau, 2.5mM; lanthanum, La³⁺, 50 µM; diphenyleneiodonium, DPI, 5 µM; none, –) on RGM of di-4-ANEPPDHQ-labelled tobacco BY-2 cells without elicitor treatment (Ctl, black histograms) or after a 5min cryptogein elicitation (Cry, 50nM, grey histograms). (B) Effect of H₂O₂ addition (100 µM) on RGM of di-4-ANEPPDHQ-labelled tobacco BY-2 cells. (C) Effect of the NADPH oxidase activity inhibitor DPI (5 µM, grey histograms, or 20 µM, black histogram) on RGM of di-4-ANEPPDHQ-labelled tobacco BY-2 cells treated with different elicitors (–, none; Cry, 50nM; OG, 50ng ml⁻¹; or Flg22, 20nM). (D) Effect of elicitors (–, none; Cry, 50nM; OG, 50ng ml⁻¹; or Flg22, 20nM) on RGM of di-4-ANEPPDHQ-labelled tobacco gp3 cells. Mean values ±SEM (n>4 independent experiments). Asterisks indicate a significant difference (P-value <0.05).

Fig. 4.

Effect of defence elicitors on PM fluidity of BY-2 cells. Tobacco BY-2 cells were treated with either oligogalacturonides (OG, black histogram) or cryptogein variants affected in their ability to load up sterols (grey histograms). After a 5min treatment, cells are labelled with di-4-ANEPPDHQ (3 µM) and half-time recovery of maximal fluorescence after photobleaching was measured for untreated (Ctl, white histogram) or elicited cells (50ng ml⁻¹ OG, or 50nM of different purified cryptogein variants: Cry X24 corresponding to the wild type form of cryptogein produced in Pichia pastoris; Cry V84F, Cry L41F, and Cry V84F/L41F corresponding to mutated forms). Mean values ±SEM (n>4 independent experiments). Asterisks denote a statistically significant difference (P-value <0.05).

Fig. 5.

Cryptogein induced an increase of PM fluidity in Arabidopsis cells. FRAP experiments were performed on di-4-ANEPPDHQ-labelled Arabidopsis suspension cells after a 5min treatment, and half-times of fluorescence recovery were reported. Control corresponds to no addition of elicitor, 5min. Mean values ±SEM (n>3 independent experiments) and an asterisk denotes a statistically significant difference (P-value <0.05).

Fig. 6.

Sterol trapping enhances ROS production induced by the elicitation signalling cascade. (A) Fluidity is enhanced by sterol depletion. Half-maximal time of fluorescence recovery was measured by FRAP experiments after sterol depletion (5min of a 50nM cryptogein elicitation, cry, or 15min of a 5mM methyl-β-cyclodextrin treatment, MβCD) and/or phosphorylation inhibition (by a 5min incubation with 2.5mM staurosporin, Stau). (B and C) Early events of the signalling cascade were measured in control conditions (no addition in the medium, –) and after elicitation treatment with either 50nM cryptogein (Cry) or 50ng ml⁻¹ oligogalacturonides (OG). A pre-incubation with (grey histogram) or without (black histogram) 5mM MβCD was performed to evaluate the effect of sterol trapping on these parameters. (B) ROS production measurement. The sum of the ROS production during the first 60min was measured by chemiluminescence and reported relative to the elicitor-induced treatment value (without sterol depletion). (C) pH alkalinization was evaluated after 30min of MβCD treatment. Mean values ±SEM (n>5 experiments). Letters indicate a significant difference between treatments (P-value <0.05).

Fig. 7.

Membrane events of the early signalling cascade induced in plant suspension cells by defence elicitors. (A) In tobacco cells, a different mode of recognition could be proposed to detect cryptogein (cry), flagellin (flg22), and oligogalacturonide (OG). Elicitor perception triggers kinase activation that leads to calcium influx. Both mechanisms are able to activate the NADPH oxidase-driven ROS burst, which in turn increases membrane order. In parallel, the sterol-trapping ability of cryptogein leads to an increase in membrane fluidity which enhances the ROS burst intensity. (B) In Arabidopsis cells, upon flg22 binding to its receptor, complex formation triggers rapid phosphorylation events. The signal transduction downstream of ligand perception includes a Ca²⁺ burst and activation of RBOH required for the ROS burst. Activation of RBOH is required for the increase of membrane order. In this cell context, cryptogein is still able to trap sterol, and concomitantly to increase membrane fluidity, without inducing any signalling pathway.