SMS treatment of A. thaliana leaves induces ROS and resistance to B. cinerea

We have treated leaves of A. thaliana by gently rubbing them between thumb and forefinger, a mechanical stress that is herewith referred to as a soft mechanical stimulation (SMS). The inoculation of leaves immediately after SMS with spores of B. cinerea led to a strong decrease in lesion size (Figure 1A). A rapid burst in ROS evidenced by a green fluorescence was observed in leaves infiltrated with 5-(and-6)-carboxy-2,7-dichlorodihydrofluorescein diacetate (DCF-DA) immediately after SMS (Figure 1B). DCF-DA detects a broad range of oxidizing reagents including H 2 O 2 and O 2 - and its use has been previously described [10]. SMS-induced resistance as well as wound-induced resistance were still detected in mutants of NADPH oxidase D (atrboh D) and F (atrboh F) as well as in the double mutant (atrboh D/F) meaning that others RBOH proteins are implicated in the formation of ROS (Additional file 1). The response to SMS showed a dose-dependence (Figure 1C-D). Applying only one event of SMS already lead to a small but detectable decrease in lesion size (Figure 1C) as well as production of discrete patches of green DCF-DA-fluorescence (Figure 1D). We used 10 stimulations throughout this study as this lead to the strongest effects. Unstimulated upper leaves of plants stimulated on one lower leaf were not protected against B. cinerea (data not shown) showing the absence of a systemic effect. Moreover, wearing latex gloves was equally effective for SMS-induced resistance (data not shown). The SMS-induced resistance to B. cinerea was transient: when plants were inoculated 8 h after SMS, about 50% of the resistance was lost and 24 h after SMS, plants were fully susceptible (Additional file 2). SMS was followed by a rapid change in intracellular calcium as detected using both yellow cameleon- or aequorin-expressing plants [16] (Figure 2A, B). The expression of so-called touch genes previously associated with mechanical stimulation such as TCH3, TCH4, CML24 and CML39[17] was also induced 0.5 to 1 h after SMS treatment (Figure 2C). The effect of SMS could still be observed in mutants of JA biosynthesis and signaling (Figure 3). The ethylene mutant ein2-1 also responds to SMS (Benikhlef, 2010; PhD thesis, University of Fribourg).

Figure 1 Resistance to B. cinerea and ROS production in response to SMS in leaves of A. thaliana Col0 . (A) Effect of 10 SMS events on resistance of A. thaliana Col0 to B. cinerea (n = 48; ±SE). Four representative pictures of necrosis caused by B. cinerea were placed above each histogram as a visual illustration. (B) Quantification of ROS production in leaves of A. thaliana Col0 after 10 events of SMS (n = 12; ±SD). ROS were determined immediately after SMS. Three representative images of the fluorescent leaf surface were placed above each histogram as a visual illustration. (C) Dose–response of SMS-induced resistance to B. cinerea and ROS accumulation in leaves of A. thaliana Col0. Single or multiple SMS events were carried out on leaves prior to inoculation with B. cinerea (n = 48; ±SE). (D) Dose–response of SMS-induced ROS accumulation in leaves of A. thaliana Col0. Single or multiple SMS events were carried out on leaves prior to quantification of ROS production (n = 12; ±SD). One representative image of the fluorescent leaf surface was placed above each histogram as a visual illustration. For all experiments in this figure, plants were kept under humid conditions after treatment and each experiment was repeated 4 times with similar results. Asterisks indicate statistically significant differences between treated samples and non-treated samples, T-Test (p < 0,01). Full size image

Figure 2 Induction of a calcium peak and touch-induced genes after SMS. (A) Changes in cytosolic calcium levels after one SMS event at time 0 s were monitored by FRET using the cameleon yellow protein YC3.6. The experiment was repeated 5 times, one representative time-course is presented. A visual illustration of the time course was placed above the curve at the indicated time point. (B) Changes in cytosolic calcium levels after five SMS events at time 0 s were monitored using the calcium sensing protein aequorin. The experiment was repeated 5 times, one representative time-course is presented. (C) The expression of selected touch genes was determined at various times after SMS (10×)(n = 3; ±SD). The experiment was performed three times with similar results. Asterisks indicate statistically significant differences between SMS-treated samples at different time point (0.5,1, 24 h) in comparison to non-treated samples (0), T-Test (p < 0,05). Full size image

Figure 3 Independence of SMS-induced resistance on jasmonic acid. Leaves of JA mutants were SMS-treated 10 times prior to inoculation with B. cinerea. SMS-induced resistance to B. cinerea was still detected in dde2.2, opr3 and coi1.16 (n = 64; ±SE), the experiment was repeated twice with similar results. After SMS, all plants were kept under humid conditions. Asterisks indicate statistically significant differences between non-treated and SMS-treated plants for Col0 and each mutant, T-Test (p < 0,01). Full size image

SMS is not accompanied by cellular damage

We next examined the occurrence of overt wounding after SMS, since wounding was shown previously to strongly affect resistance to B. cinerea[7]. Macroscopic signs of wounding were not visible on SMS-treated leaves. Nevertheless we assessed the effect of SMS on the cell surface by scanning electron microscopy (SEM) of live leaf surfaces as well as vital staining using trypan blue. SMS-treated epidermal cells looked perfectly turgid when viewed under the SEM (Figure 4). The waxy surface of some cells appeared slightly affected but the cells themselves retained turgidity. Some trichomes and cells at their base displayed damage (Figure 4B). When SMS-treated leaves were stained using trypan blue, a vital dye that marks the presence of dead cells, epidermal cells were essentially intact 1 h after treatment with the exception of isolated cell groups that stained in blue at the basis of trichomes (Figure 5A), in agreement with observations made using the SEM directly after SMS. Thus, SMS did not lead to massive cellular damage compared to wounding with forceps that was observed previously [10]. The question remains if the damaged cells at the basis of the trichomes might constitute a sufficiently strong wound stimulus to induce ROS and resistance. This question was approached using the trichome-less glabrous gl1 mutant of Arabidopsis [18]. SEM images of leaf surfaces of the gl1 mutant were compared to WT plants and in both plants cells retained turgidity after SMS (Figure 4). No damaged cells were observed using the vital stain trypan blue in gl1 mutant after SMS (Figure 5A). In fact, the untreated gl1 mutant is as susceptible to B. cinerea as WT plants and after SMS, gl1 displayed resistance to B. cinerea to the same extent as the wild type (Figure 5B). Thus, SMS induced resistance independently of the presence of trichomes and SMS-induced resistance to B. cinerea is not based on wounding of cells.

Figure 4 Integrity of epidermal cells after SMS treatment visualised by SEM. (A) Surfaces of leaves of A. thaliana Col0 were observed by SEM after SMS (10×). (B) Damage to trichomes after SMS (10×) compared to non-treated leaves. (C) Surfaces of leaves of the glabrous mutant gl1 of A. thaliana Col0 observed by SEM after SMS (10×). All observations of this figure were repeated 25 times on 5 different leaves. Representative samples are shown. Full size image

Figure 5 Integrity of epidermal cells after SMS treatment visualised by vital staining. (A) Leaves of A. thaliana Col0 or the glabrous gl1 mutant were treated by SMS (10X) or wounded with forceps and visualized after 1 day by using Trypan blue staining. All plants were kept under humid conditions after treatment prior to trypan blue staining. Observations were made 2 times on 6 different leaves. Representative samples are shown. (B) Resistance to B. cinerea in response to SMS in gl1 mutant compared to Col0 plants (n = 96; ±SE). After B. cinerea inoculation, all plants were kept under humid conditions. The experiment was repeated 2 times. Asterisks indicate statistically significant differences between non-treated leaves and SMS-treated leaves, T-Test (p < 0,01). Full size image

Alterations in cuticular permeability and SMS

An accumulation of ROS was previously observed in A. thaliana plants displaying increased cuticular permeability such as mutants defective in cuticle biosynthesis or in the formation of abscisic acid (ABA) [10]. Therefore we have determined if the SMS-stimulated leaves underwent a change in cuticular permeability. The permeability of the cuticle of SMS-stimulated leaves was assessed using various diagnostic tests. SMS-treated leaves displayed an increase in cuticular permeability as collectively indicated by increased chlorophyll leakage, retention of toluidine blue as well as Calcofluor white staining (Figure 6A to C).

Figure 6 The effect of SMS on the permeability of A. thaliana Col0 leaves. (A) Permeability of the cuticle measured by chlorophyll leaching in SMS-treated leaves compared to controls (n = 3; ±SD); the experiment was carried out 2 times, one typical result is represented. (B) Droplets of toluidine blue were placed on the both sides of leaf surface for 2 h in high humidity then the leaf surface was rinsed with water. The blue stain that remains attached to the cell wall is indicative of a permeable cuticle. The percentage of droplets stained in blue relative to the total inoculated droplets was calculated (n = 3; 90 droplets per experiment; ±SD). Two representative pictures of leaves stained with toluidine blue were placed above each histogram as a visual illustration. Asterisks indicate statistically significant differences between SMS-treated and non-treated Col0 leaves, T-Test (p < 0,01). (C) leaves were bleached overnight in ethanol then stained with Calcofluor white that binds to cellulose, and viewed under UV light. Calcofluor staining to the leaf is indicative of a permeable cuticle (the experiment was carried out 3 times, one typical result is represented). Full size image

SMS is not accompanied by changes in ABA levels

Wound-induced resistance to B. cinerea is lost when wounded plants are not maintained under a humid environment (in covered trays). This loss is caused by ABA, the level of which increases under dry conditions (trays uncovered) [10]. In accordance, mutants impaired in ABA were fully resistant after wounding, both when plants were maintained under humid or dry environments [10]. Interestingly, increase in resistance and production in ROS were observed whether plants were maintained at humid or dry conditions after SMS (Figure 7A and B). Consequently, we also determined possible changes in the level of ABA. No changes were observed between the levels of ABA after SMS in plants maintained under humid or dry conditions (Additional file 3). This marks a clear difference between SMS and wound-induced resistance.

Figure 7 The effect of humidity on resistance of A. thaliana Col0 to B. cinerea and ROS accumulation after SMS. Leaves were stimulated by SMS and maintained for 1.5 h under high humidity in tightly covered well-watered trays (humid) or left in uncovered trays (dry) at room conditions prior to inoculation with B. cinerea and ROS detection. (A) Resistance to B. cinerea (n = 64; ±SE). One representative picture of the growth of B. cinerea was placed above each histogram as a visual illustration (trypan blue staining was carried out 3 days after inoculation). (B) Quantification of ROS production; three representative images of the fluorescent leaf surface were placed above each histogram as a visual illustration (n = 16; ±SD). Asterisks indicate statistically significant differences between treated samples and non-treated samples in humid and dry conditions, T-Test (p < 0,01). Full size image

SMS and the surface wax layer

Since SMS likely perturbs the waxy surface of the plant without much damage to the underlying cells (Figures 4 and 5A), we explored the importance of the wax layer on the leaf surface. We have used the myb96-1 mutant affected in the transcription factor MYB96, involved in the biosynthesis enzymes condensing very-long-chain fatty acids involved in cuticular wax biosynthesis [19]. Untreated myb96-1 mutants displayed increased resistance to B. cinerea (Figure 8A). The ROS response of myb96-1 mutants to B. cinerea was much faster than in the wild type since green fluorescence was detected already 3 hours after inoculation with B. cinerea (Figure 8B). The myb96-1 mutant also displayed a slight modification of permeability as indicated by the toluidine blue and Calcofluor white test, but this modification was not detected with the chlorophyll leakage test (Figure 8C-E). Thus, myb96-1 altered in the wax layer displays a somewhat similar syndrome (increased resistance, ROS and a partial increase of permeability) although less obvious that the wild type leaves after SMS treatment.

Figure 8 Resistance to B. cinerea and ROS production in waxless myb96KO mutants. (A) Resistance to B. cinerea in myb96-1 mutant and wild type Col0 plants. Four representative pictures of necrosis due to B. cinerea are illustrated above each histogram. After B. cinerea inoculation, all plants were kept under humid conditions (n = 128; ±SE). The experiment was repeated twice with similar results. Asterisks indicate statistically significant differences between Col0 and myb96-1 mutant, T-Test (p < 0,01). (B) Quantification of ROS production at 3, 6, 10 h after inoculation with B. cinerea (Bc) or mock treatment in myb96-1 mutants compared to Col0. After treatment, all plants were kept under humid conditions (n = 6; ±SD). Asterisks indicate statistically significant differences between Col0 and myb96-1 mutant after B. cinerea inoculation or mock treatment, T-Test (p < 0,01). (C) Permeability of the cuticle in WT Col0 leaves compared to myb96-1 mutant. Leaves were placed in ethanol and the release of chlorophyll was followed over time (n = 3; ±SD). The experiment was carried out 3 times, one typical result is represented. (D) Permeability of the cuticle as determined by the toluidine blue test. The blue stain that remains attached to the cell wall is indicative of a permeable cuticle. The percentage of droplets stained in blue relative to the total inoculated droplets was calculated (n = 3; 90 droplets per experiment; ±SD). Two representative pictures of leaves stained with toluidine blue were placed above each histogram as a visual illustration. Asterisks indicate statistically significant differences between Col0 and myb96-1 mutant, T-Test (p < 0,01). (E) Permeability of the cuticle as determined after Calcofluor white staining and viewing under UV light. Calcofluor retention by the cellulose is indicative of a permeabilized cuticle (the experiment was carried out 3 times, one typical result is represented). Full size image

SMS and leaf diffusates

Changes in the permeability of the cuticle were shown to be associated with the leakage of diffusates that prevent the development of B. cinerea in vitro and in vivo[15, 20]. We have tested if bioactive diffusates can be obtained from leaf surfaces of SMS-treated WT or myb96-1 plants. SMS applied to gl1 and myb96-1 mutants was similarly effective as on SMS-treated WT plants (Figure 9). Without SMS, diffusates collected from the surfaces of both gl1 and myb96-1 plants were inactive similarly to those from WT plants. Thus SMS acted on leaf surfaces in a comparable way in plants or mutants displaying increased cuticular permeability [15, 20].