Tetiana Kalachova
email: kalachova@ueb.cas.cz

Romana Pospichalova

Petr Marsik

Pavel Kloucek

Olga Valentová

Eric Ruelland

Hana Leontovyčová

Oksana Iakovenko

Petr Maršík

Pavel Klouček

Romana Pospíchalová

Martin Janda

Daniela Kocourková

Jan Martinec

Lenka Burketová

Interplay between phosphoinositides and actin cytoskeleton in the regulation of immunity related responses in Arabidopsis thaliana seedlings

Keywords: Actin filaments, callose and phosphatidylinositol-4-kinase mutants Actin, Latrunculin B, Salicylic acid, Callose, Phosphatidylinositol-4-kinase

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INTRODUCTION

Plants are always confronted to changes in their environment. This can concern physical parameters such as light intensity and/or quality, temperature or water availability, or interactions with other organisms, including bacteria or fungi. Some of the interactions can lead to diseases.

Nowadays, plant responses to the environmental challenges, either abiotic or biotic, are an object of an intense research. A novel aspect of such a research is the investigation of the roles of processes that were so far mainly (or uniquely) considered as important for the cell structure. Such a process is the dynamics of cytoskeleton. Cytoskeleton is comprised of microfilaments and microtubules. Actin filaments (F-actin) are highly dynamic structures formed from monomers (Gactin) [START_REF] Drobak | The Role of the Actin Cytoskeleton in Plant Cell Signaling[END_REF]. Even in basal conditions, filaments constantly grow at one end and depolymerize at the opposite one [START_REF] Li | Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity[END_REF]. The filaments are indispensable for cell shaping, growth and polarity [START_REF] Andreeva | Inhibition of phospholipase C disrupts cytoskeletal organization and gravitropic growth in <i>Arabidopsis</i> roots[END_REF], cytokinesis [START_REF] Brill | A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis[END_REF], but also for intracellular trafficking of vesicles [START_REF] Geitmann | Navigating the plant cell: intracellular transport logistics in the green kingdom[END_REF]. Yet, the direct mechanisms regulating actin dynamics in regards to vesicle trafficking are still waiting to be revealed. A particular role has been proposed for phospholipids, especially phosphoinositides [START_REF] Ebine | Interaction of the Arabidopsis GTPase RabA4c with Its Effector PMR4 Results in Complete Penetration Resistance to Powdery Mildew[END_REF].

Phosphoinositides are phosphorylated forms of phosphatidylinositol (PI). From the position and number of the phosphate groups on the inositol ring, we can distinguish PI-3-phosphate (PI3P), PI-4-phosphate (PI4P) and PI-4,5-bisphosphate (PIP2). Phosphoinositides play a key role in vesicle trafficking [START_REF] Delage | Signal transduction pathways involving phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: Convergences and divergences among eukaryotic kingdoms[END_REF]) and also interact with cytoskeleton: for instance, F-actin capping protein interaction with actin monomers is regulated by PIP2 [START_REF] Pleskot | Structural Insights into the Inhibition of Actin-Capping Protein by Interactions with Phosphatidic Acid and Phosphatidylinositol (4,5)-Bisphosphate[END_REF]). This makes phosphoinositides potential central players in the regulation of cytoskeleton dynamics versus vesicle trafficking. Phosphatidylinositol-4 kinases (PI4Ks) are the first enzymes committing PI into phosphoinositides phosphorylated at position 4 of inositol. The study of these enzymes and of their corresponding mutants can be useful to investigate the regulation of the vesicle trafficking together with cytoskeleton dynamics. In Arabidopsis thaliana, the isoforms AtPI4Kβ1 and AtPI4Kβ2 appear to be located in the endomembrane system [START_REF] Antignani | Recruitment of PLANT U-BOX13 and the PI4Kβ1/β2 Phosphatidylinositol-4 Kinases by the Small GTPase RabA4B Plays Important Roles during Salicylic Acid-Mediated Plant Defense Signaling in Arabidopsis[END_REF][START_REF] Kang | Electron Tomography of RabA4b-and PI-4Kβ1-Labeled Trans Golgi Network Compartments in Arabidopsis[END_REF]. They participate in cytokinesis in A.thaliana roots by targeting actin filaments to the cell plate [START_REF] Lin | A dual role for cell plate-associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis[END_REF].

Regarding cytoskeleton and the responses to pathogens, it must be noted that soon after contact between a plant cell and a pathogen, actin filaments get rapidly reordered to support transport of the stress-related compounds. For instance, in the case of fungal infection, actin is needed for the transport of antimicrobial compounds and callose to the penetration site [START_REF] Hardham | Cell biology of plant-oomycete interactions[END_REF]. This results in forming cell wall appositions that arrest pathogen growth at the invaded cell [START_REF] Sassmann | An Immune-Responsive Cytoskeletal-Plasma Membrane Feedback Loop in Plants[END_REF]. This is part of plant immunity response. In case of bacterial infection, the reaction at the level of cytoskeleton is more diffuse along the leaf tissue but an increase of actin filament density and bundling can nevertheless be measured [START_REF] Lu | Quantitative Evaluation of Plant Actin Cytoskeletal Organization During Immune Signaling[END_REF]. Various components of pathogen cells can trigger actin polymerization in plants. The most described are the so-called microbe associated molecular patterns (MAMPs), like flagellin flg22, elf18, chitin, oligogalacturonides [START_REF] Li | Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity[END_REF], or secreted effectors (e.g. HopG1 of Pseudomonas syringae, [START_REF] Shimono | The Pseudomonas syringae Type III Effector HopG1 Induces Actin Remodeling to Promote Symptom Development and Susceptibility during Infection[END_REF]. Those compounds not only affect cytoskeleton, but induce other immune responses, especially reactive oxygen species formation, defence genes expression, biosynthesis of salicylic acid (SA) [START_REF] Bigeard | Signaling Mechanisms in Pattern-Triggered Immunity (PTI)[END_REF].

A well described response to pathogens is callose accumulation [START_REF] Schneider | Cellulose and callose synthesis and organization in focus, what's new?[END_REF].

Callose is a polysaccharide, β-1,3-glucan, synthetized in the apoplast and involved in various processes in plants including cytokinesis (Chen et [START_REF] Keppler | 3-Aminobenzamide Blocks MAMP-Induced Callose Deposition Independently of Its Poly(ADPribosyl)ation Inhibiting Activity[END_REF]. We therefore wanted to investigate the influence of actin depolymerization on callose accumulation. An elegant way to investigate the role of cytoskeleton is the use of drugs that alter cytoskeletal dynamics. Latrunculin B (latB) is such a drug. It originates from a Red Sea sponge Latrunculia magnificans. LatB reversely binds actin monomers preventing their polymerization [START_REF] Morton | Latrunculin alters the actin-monomer subunit interface to prevent polymerization[END_REF]. It is often used in plant biology to assess the role of actin in endo-and exocytosis [START_REF] Moscatelli | Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes[END_REF], pollen tube elongation [START_REF] Gibbon | Latrunculin B has different effects on pollen germination and tube growth[END_REF] and growth [START_REF] Baluška | Latrunculin B-Induced Plant Dwarfism: Plant Cell Elongation Is F-Actin-Dependent[END_REF]. Concerning the response to pathogens, it has been shown that when applied simultaneously with pathogens, latB negatively affects plant resistance to them (Henty-Ridilla et al, 2013). However, latB is able to activate SA-associated gene expression in Arabidopsis thaliana [START_REF] Matoušková | Changes in actin dynamics are involved in salicylic acid signaling pathway[END_REF]. In Brassica napus and Nicotiana benthamiana, the pre-treatment of plant leaves with low concentrations of latBincreased SA levels in tissues, activated defence gene expression and was sufficient to increase resistance to further infection. Similar results were obtained with another actin disrupting drug, cytochalasin E [START_REF] Leontovyčova | Actin depolymerization is able to increase plant resistance against pathogens via activation of salicylic acid signalling pathway. Scientific Reports in press 32[END_REF].

We firstly show that actin disruption by latB triggers callose accumulation in A. thaliana seedlings through the callose synthase PMR4. Using mutants altered in SA signaling (pad4), biosynthesis (sid2) and accumulation (NahG), we demonstrate that this callose deposition is SAindependent. Furthermore, we found that latB effect on gene expression does not correspond to a general transcriptome deregulation, but that specific pathways related to the response to pathogens are the ones triggered by latB. Amongst the gene responses, we were also able to reveal SAdependent and SA-independent effects of latB. Interestingly, we show that deficiency in PI4Kβ1β2 enhanced the susceptibility of actin filaments to latB while latB does not appear to impact phosphoinositides levels on plasma membrane. To study the role of PI4Kβ1β2 in SA-independent responses to actin disruption, we have introduced NahG construct as well as sid2 and pad4 mutations into pi4kβ1β2 background. Surprisingly, the latB treatment still strongly induced SA in pad4pi4kβ1β2 and NahGpi4kβ1β2 triple mutants, while SA accumulation was prevented in pad4 and NahG mutant plants. To conclude, here we characterize the immunity-like responses to latB, showing that actin depolymerisation acts through both SA-dependent and SA-independent pathways. Moreover, we reveal the existence of an unexpected cross-talk between actin filaments and phosphoinositides in the regulation of those defence-related responses.

Materials and methods

Plant material

The following Arabidopsis thaliana genotypes were used: Columbia-0 (WT); NahG [START_REF] Delaney | A central role of salicylic acid in plant disease resistance[END_REF]; NahGpi4kβ1β2 (Šašek et al, 2014); pad4.1 (NASC stock N3806); pad4pi4kβ1β2 (this study); sid2, pi4kß1ß2, sid2pi4kß1ß2 [START_REF] Kalachova | Identification of salicylic acidindependent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant[END_REF]; pmr4; pUBQ::PMR4-GFP in WT background; pUBQ::PMR4-GFP in pmr4 background [START_REF] Kulich | Exocyst Subunit EXO70H4 Has a Specific Role in Callose Synthase Secretion and Silica Accumulation[END_REF]; Lifeact-GFP [START_REF] Cvrčková | A new kymogram-based method reveals unexpected effects of marker protein expression and spatial anisotropy of cytoskeletal dynamics in plant cell cortex[END_REF]; pUBQ::mCitrine-2xFAPP1 [START_REF] Simon | A multi-colour/multi-affinity marker set to visualize phosphoinositide dynamics in Arabidopsis[END_REF].

Plant cultivation and treatment

Seedlings were grown in MS medium, containing 4.41 g Murashige and Skoog medium including vitamins (Duchefa, Netherlands) and 5 g sucrose per litre, pH adjusted to 5.7 using 1M KOH.

Surface-sterilized seeds were sown in 24-well plates, 4-5 seeds per well containing 400 μL of medium and cultivated for 10 days before treatment under a short-day photoperiod (10 h/14 h light/dark regime) at 100-130 μE m -2 s -1 and 22°C. On the 7th day, 200 μL of fresh medium was added to the wells. Latrunculin B (latB, Sigma-Aldrich, USA) was dissolved in DMSO at 2 mM stock solution. The seedlings were treated 24 h with 200 nM latB and 0.01% DMSO as mock control prior to sampling.

Gene expression analysis

The whole seedlings (approximatively 300 µg fresh weight) from three wells were immediately frozen in liquid nitrogen. The tissue was homogenized in plastic Eppendorf tubes with 1 g of 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, USA). Total RNA was isolated using Spectrum Plant Total RNA kit (Sigma-Aldrich, USA) and treated with a DNA-free kit (Ambion, USA). Subsequently, 1 μg of RNA was converted into cDNA with M-MLV RNase H - Point Mutant reverse transcriptase (Promega Corp., USA) and an anchored oligo dT21 primer (Metabion, Germany). Gene expression was quantified by qPCR using a LightCycler 480 SYBR Green I Master kit and LightCycler 480 (Roche, Switzerland). The PCR conditions were 95 °C for 10 min followed by 45 cycles of 95 °C for 10 s, 55 °C for 20 s, and 72 °C for 20 s. Melting curve analysis was then conducted. Relative expression was normalized to the housekeeping gene AtTIP41. A list of the analysed genes and primers is available in Supplemental table ST1.

Trypan blue and anilin blue staining

Seedlings were immersed into the staining solution (10 mL lactic acid (85%, w:w), 10 mL phenol, 10 mL glycerol, 10 mL distilled H2O, 40 mg trypan blue) for 2 min (Fernández-Bautista et al, 2016). Seedlings were then washed twice in ethanol:glacial acetic acid (1:3, v:v) until leaves were fully decoloured. Seedlings were rehydrated by incubating in ethanol solutions of decreasing concentrations (70%, 50%, 30%, v:v) for at least 30 min each. They were then stained in 0.01 % aniline blue in 150 mM K2HPO4, pH 9.5 for 4h and kept in water for the microscopy observation.

Callose imaging and quantification

Callose deposition was observed by fluorescence microscope using a Zeiss AxioImager ApoTome2 (objective 5x). Callose accumulation was calculated using FIJI software (https://fiji.sc/) [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] as the percent occupancy of aniline blue signal (spots) per region of interest (ROI). One round-shaped ROI (d=1500 µm) was taken from one cotyledon, at least 18 independent cotyledons were analysed per variant.

Confocal microscopy

For in vivo microscopy, a Zeiss LSM 880 inverted confocal laser scanning microscope (Carl Zeiss AG, Germany) was used with either a 40× C-Apochromat objective (NA = 1.2 W) or a 20x Plan-Apochromat objective (NA = 0.8). Fluorescence associated with actin filaments (LifeAct-GFP) or phosphoinositides (mCitrine-2xFAPP1) was acquired at excitation 488 nm emission 490-540 nm for GFP and excitation 516nm emission 520-552 nm for mCitrine. Images were acquired at in zstacks 20µm thickness). Actin filaments density analysis was calculated by Fiji software as the percent occupancy of GFP/mCitrine signal in each Maximum intensity projection. For Lifeact-GFP, image threshold was set to include all actin filaments and area fraction was measured. For each variant, fluorescent intensity of at least 30 ROI (50x50µm) from 10-14 leaves was analysed.

Salicylic acid measurement

All seedlings from 3 wells were sampled as 1 sample, frozen in nitrogen and stored at -80°C until the analysis. Samples were homogenized in tubes with 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, CA, United States) with 2 x 500 µl of extraction reagent methanol/water/formic acid (15:4:1, v:v:v) supplemented with stable isotope-labelled 13C-SA internal standards, each at 100 ng per sample, to check the recovery during the purification and to validate the quantification. Extract was subjected to solid phase extraction using Oasis MCX cartridges (Waters Co., Milford, MA, United States) and eluted with methanol. The eluate was evaporated to dryness and dissolved in 30 μl of 15% acetonitrile/water (v/v) immediately before the analysis. immediately before the analysis. Quantification was performed on an Ultimate 3000 high-performance liquid chromatograph (UHPLC, Dionex; Thermo Fisher Scientific, Waltham , MA, United States) coupled to IMPACT II Q-TOF ultra-high resolution and high mass accuracy mass spectrometer (HRAM-MS; Bruker Daltonik, Bremen, Germany). Separation was carried out using column Acclaim RSLC 120 C18 (2.2 m, 2.1x100 mm; Thermo Fisher Scientific, Waltham, MA, United States) tempered at 30°C and mobile phase consisting of 0.1% formic acid (A) and methanol (B) by gradient elution. The gradient started at 1%B and going to 39 % B in 3min, 60 % B in 7 min, 100 % B in 8 min, followed by 3 min of washing by 100% B and 5 min equilibration at initial conditions (1%B). Flow rate was changing during separation from 200 l/min to 300 l/min in 7 min and then kept at this rate to 16 min. Injection volume was 5 L. The full-scan data were recorded in negative electrospray ionization (ESI) mode and processed using Data Analysis 4.3 and TASQ 1.4 software (both Bruker Daltonik, Bremen, Germany). SA and labelled internal standard ( 13 C6-SA) were monitored as ions of their deprotonated molecules [M-H]-(137.0239 m/z and 143.0440 m/z, respectively). SA content was expressed in ng/g fresh weight (FW).

RESULTS

Latrunculin B induces callose via PMR4 callose synthase

Ten-day-old A. thaliana seedlings were treated for 24 h with 200 nM latB. As expected, latB treatment caused actin disruption, measured as the decrease of filament abundancy (fig. S1).

The concentration used for the drug did not lead to cell death (fig. S1). More unexpectedly, the treatment also caused callose accumulation (measured as the area occupied with callose spots) (fig. 1A,B). Among twelve known callose synthases in A. thaliana, PMR4 was shown to be a major one involved to biotiс stress responses [START_REF] Nishimura | Loss of a Callose Synthase Results in Salicylic Acid-Dependent Disease Resistance[END_REF][START_REF] Kulich | Exocyst Subunit EXO70H4 Has a Specific Role in Callose Synthase Secretion and Silica Accumulation[END_REF]. We therefore tested its contribution to latB-triggered callose. In the pmr4 mutant seedlings, the callose level detected after latB treatment was nearly equal to that in mock-treated control. When a pUBQ::PMR4-GFP construct was expressed in WT or pmr4 backgrounds, basal callose level in cotyledons remained low. Yet, when latB was added, callose accumulation was restored (Fig. 1B).

Besides, such an induction did not rely on transcriptional changes, as soon as the expression of PMR4 was not affected (Fig. 1C).

Latrunculin B effects on callose accumulation is SA-independent

The fact that latB treatment causes callose accumulation through PMR4 strongly suggests that other defence-related responses should be studied. We therefore decided to study the role of SA content and signaling pathway in the observed callose deposition. To do so, we first measured SA levels in seedlings after latB treatment. A 20-fold increase of SA was observed in WT seedlings. Notably, pmr4 plants behaved similarly to WT, suggesting callose to be either downstream, either independent on SA (fig. 1D, Supplementary table ST3). To address this, we analyzed SA induction and callose deposition in plants with altered SA pathway: NahG mutant plants, expressing the bacterial salicylate hydroxylase that degrades SA to inactive catechol and therefore unable to accumulate SA; sid2 plants, deficient in ISOCHORISMATE SYNTHASE 1 (ICS1); and pad4 plants, with a mutation in PHYTOALEXIN DEFICIENT 4 (PAD4) (fig. 2, Supplementary table ST3). PAD4 protein has been shown to regulate the synthesis of SA via the ICS1 pathway [START_REF] Cui | A core function of EDS1 with PAD4 is to protect the salicylic acid defense sector in Arabidopsis immunity[END_REF]. As expected, both NahG and pad4 plants had lower basal content of SA, and smaller (approx. 5-fold) increase in SA after latB comparing to WT. sid2 plants showed no increase in SA, pointing out that the detected SA was produced by ICS1. On the contrary, callose production was not affected any of the studied mutants, confirming SA-independent nature of the process (fig. 2,B,C).

Latrunculin B affects gene expression in both SA-dependent and -independent manner

We had already shown that latB could induce the expression of some genes related to plant defence [START_REF] Leontovyčova | Actin depolymerization is able to increase plant resistance against pathogens via activation of salicylic acid signalling pathway. Scientific Reports in press 32[END_REF][START_REF] Matoušková | Changes in actin dynamics are involved in salicylic acid signaling pathway[END_REF]). Here we studied whether intracellular level of SA or an intact SA-signaling pathway is needed for latB to trigger gene expression changes (figure 3, Supplementary figure S2, Supplementary table ST2). To do so, we analysed the expression of several defence marker genes in NahG and pad4 mutants. Eleven-day-old in vitro grown plants (whole seedlings) of WT and mutant genotypes were harvested for gene expression studies 24 hpt by 200nM latB. Concerning the gene response in WT plants, we show here that the defence-associated PR1, PR2, WRKY38, ICS1 and PAD4 were induced by latB (Figure 3). We also detected an activation of the wounding marker BAP1, even though no cell death was yet detected by trypan blue staining (Supplemental figure S1). On the contrary, no significant changes were observed in the expression of SA biosynthetic genes other than ICS1 (ICS2, PAL1, PAL2, PAL3).

To be noted, ICS2 expression seems to be very dependent on the growth conditions, as we previously detected its induction after latB treatment [START_REF] Leontovyčova | Actin depolymerization is able to increase plant resistance against pathogens via activation of salicylic acid signalling pathway. Scientific Reports in press 32[END_REF]. In addition, neither jasmonate biosynthesis marker genes (LOX2, AOS1), nor jasmonate responsive genes (VSP2 and PDF1.2), nor ethylene marker gene (ERF1) were significantly affected by the treatment (Supplemental figure S2, Supplemental Table ST2). However, we detected that PR1 and WRKY38 inductions by latB were abolished in NahG and pad4 mutants, strongly suggesting these inductions rely on a SA-dependent pathway (fig. 3). This also involves that SA is accumulated in response to latB (Fig. 2 A). On the contrary, the expression of PR2 in response to latB appears not to be dependent on SA since it was still present in NahG and pad4 mutants. Similarly, the expression of the PAD4 by latB was still present in NahG plants: it does not rely on SA accumulation. The expression of ICS1 in response to latB does not appear to be controlled by SA level in the tissues, as seen in NahG mutant. Yet, PAD4 protein regulates ICS1 expression, at least partly. As for the induction of the expression of BAP1 by latB, it was diminished in NahG and pad4 mutants (though still present), and so can be considered as partly SA-dependent.

We can thus conclude that latB induces several defence-like responses: there is an induction of SA; it leads to the induction of PR1 and WRKY38. However, an alternative, SAindependent, pathway also exists. It controls the expression of PR2, ICS1, PAD4 and callose accumulation in response to latB.

Effects of latB on cytoskeleton are enhanced in pi4kß1ß2 double mutant

Phosphoinositides, that are the phosphorylated forms of phosphatidylinositol (PI), are involved in vesicular trafficking. This process requires correct actin cytoskeleton organization [START_REF] Pleskot | When fat is not bad: the regulation of actin dynamics by phospholipid signaling molecules[END_REF]. There is therefore a link between actin cytoskeleton and phosphoinositides.

We wanted to investigate whether the effect of latB could be impacted by a modification in phosphoinositide metabolism. To do so, we used the pi4kß1ß2 double mutant. This mutant is invalidated in the two beta isoforms of phosphatidylinositol-4-kinases. This double mutant was shown to have impaired vesicle trafficking, dwarf phenotype and increased SA level (Kang et al,

2011; Šašek et al, 2014).

We firstly introduced LifeAct-GFP construct into pi4kß1ß2 background by crossing. Four independent lines were tested. No major defects in actin filaments organization in epidermal cells were observed while compared to WT (fig. 4). After 24h of latB treatment, the density of the filaments in pi4kß1ß2 plants was 20-times lower than that in non-treated double mutant, while in the WT background this decrease was 5-fold. Consequently, in pi4kß1ß2 plants the actin filaments were almost lost while in WT their occupancy was still 7% (fig 4B). The actin cytoskeleton of pi4kß1ß2 mutants thus appear to be more sensitive to latB. Meanwhile, we wanted to evaluate the impact of latB directly on the PI4P content using genetically encoded biosensor mCitrine-2xFAPP1 for PI4P [START_REF] Simon | A multi-colour/multi-affinity marker set to visualize phosphoinositide dynamics in Arabidopsis[END_REF]. The sensor mostly labels PI4P localized in plasma membrane. In our setup, the content of PI4P remained stable in response to latB (fig. S3 A,C).

Effects of latB on callose accumulation in pi4kß1ß2 double mutant is still SA-independent

Because it seems there was a positive (enhancing) interaction between latB and PI4Kbeta mutations on actin filaments, we studied SA levels, callose accumulation and gene expression responses to latB in pi4kß1ß2 plants. Indeed, latB triggered SA increase in pi4kß1ß2 plants (fig. 5A). Surprisingly, introduction of neither NahG construct nor pad4 mutation into pi4kß1ß2 background did not fully prevent SA increase as it did in WT background (fig. 2A), even though such an increase was still much lower than in WT or pi4kß1ß2. However, sid2 mutation was sufficient to block SA production in both WT and pi4kß1ß2, confirming that the entire SA pool induced by latB treatment is synthetized due to ICS1 activity in both backgrounds. As for callose, its basal levels in 8-, 10-and 14-day-old pi4kß1ß2 seedlings were similar to that of WT (Supplemental figure S4). To study the involvement of SA, the callose accumulation was also tested in triple mutants NahGpi4kß1ß2 (Šašek et al, 2014), sid2pi4kß1ß2 [START_REF] Kalachova | Identification of salicylic acidindependent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant[END_REF] and pad4pi4kß1ß2, obtained by crossing pi4kß1ß2 to pad4 (homozygous F3 plants were used).

After latB treatment, accumulation of callose in all studied mutants was similar to that in WT (fig. 5B), suggesting that callose response to latB in pi4kß1ß2 is still not SA-dependent (fig. 5B). This is consistent with our findings in WT plants.

Effects of latrunculin B on defence genes expression in pi4kß1ß2 double mutant

As for PR1 and WRKY38, after latB treatment, their induction in pi4kß1ß2 was comparable to that in WT. Yet, contrary to what was observed in WT background, their induction was also observed in NahGpi4kß1ß2 and pad4pi4kß1ß2 triple mutants (fig. 6). This is in agreement with the fact that SA is still accumulated in these triple mutants. BAP1 expression in PI4K deficient mutants after latB treatment was induced less than in WT and was partly SA-dependent (fig. 6).

Besides, neither induction of SA biosynthetic enzymes other than ICS1 was detected, nor of JA-, ABA-, or ethylene markers (Supplemental figure S5, supplemental table ST2). Reference gene TIP41.

DISCUSSION

We show here that latrunculin B induces callose accumulation in seedlings. It is also true for cytochalasin E, another actin filament disruptor (data not shown). Therefore, it is very likely that this effect of latB is due to actin depolymerization. Callose deposition was reported in responses to various stresses, like wounding or pathogen infection [START_REF] Jacobs | An Arabidopsis Callose Synthase, GSL5, Is Required for Wound and Papillary Callose Formation[END_REF]. We had previously shown that chemical disruption of actin led to some immunity-related responses such as activation of PR1 expression (Matouskova et al., 2014). In this regard, callose is particularly important, as another biotic-stress-like response, partly connected with SA. Arabidopsis genome encodes twelve callose synthases [START_REF] Voigt | Callose-mediated resistance to pathogenic intruders in plant defense-related papillae[END_REF]. PMR4 (POWLDERY MILDEW RESISTANT 4, also called GLUCAN SYNTHASE LIKE 5, GLS5 or CALS12) is precisely the one activated during infection [START_REF] Nishimura | Loss of a Callose Synthase Results in Salicylic Acid-Dependent Disease Resistance[END_REF]). Here we first show that callose accumulation induced by latB fully relies on PMR4. It indicates its connection with a biotic stress. Interestingly, using NahG and pad4 mutants, we show that latrunculin B effects on callose accumulation is SA-independent and not downstream of PAD4. To be noted, pmr4 mutant itself was reported to have a constitutively activated SA pathway in adult plants and contribute to most of callose accumulations in studies connected with biotics stres [START_REF] Nishimura | Loss of a Callose Synthase Results in Salicylic Acid-Dependent Disease Resistance[END_REF]. Nevertheless, SA level in pmr4 seedlings did not differ from that in WT (Supplementary table ST3). It was also true for pi4kß1ß2 plants, known as SA and callose accumulators while being grown up to 4-weeks [START_REF] Kalachova | Identification of salicylic acidindependent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant[END_REF]. However, neither SA nor callose levels were increased in non-stimulated seedlings (figure 5). A negative interaction between actin polymerisation, callose and SA levels has been suggested recently: natural polymorphism of genetic disruption of Actin Related Protein Complex 4 (ARPC4) affected pathogen-and wounding-induced callose deposition, as well as affected expression of PR1 in response to Sclerotinia sclerotiorum [START_REF] Badet | Expression polymorphism at the ARPC4 locus links the actin cytoskeleton with quantitative disease resistance to Sclerotinia sclerotiorum in Arabidopsis thaliana[END_REF].

As latB action on callose appeared as SA-independent, we wanted to check whether the effect of latB on gene expression was also independent on SA. We first better detailed the changes in gene expression triggered by latB. We show here that PR1, PR2, WRKY38, ICS1 and PAD4 were induced by latB in WT. It is also the case for a marker of cell death in pathogen and wounding responses, BAP1 [START_REF] Yang | The Arabidopsis BAP1 and BAP2 genes are general inhibitors of programmed cell death[END_REF]. BAP1 was described as wounding marker, but also as a SA-associated gene: it negatively regulates cell death in response to Pseudomonas syringae pv tomato and Hyaloperonospora parasitica acting in the same pathway with BON1, SNC1 and PAD4 [START_REF] Yang | The C2 domain protein BAP1 negatively regulates defense responses in Arabidopsis[END_REF]) BAP1 expression can be induced by high temperature or ROS formation, however this induction was abolished in mutants with altered SA pathways (NahG, pad4) [START_REF] Hedtmann | The Plant Immunity Regulating F-Box Protein CPR1 Supports Plastid Function in Absence of Pathogens[END_REF][START_REF] Zhu | Induction of BAP1 by a moderate decrease in temperature is mediated by ICE1 in Arabidopsis[END_REF]. In our setup, the expression of BAP1 was increased independently of the SA content in plants, indicating a new role of this gene in plant stress responses. Yet, no significant changes were observed in the expression of SA biosynthetic genes other than ICS1 (ICS2, PAL1, PAL2, PAL3), nor in the expression of LOX2 and AOS, a JA-biosynthesis marker genes, nor to the jasmonate responsive genes VSP2 and PDF1.2. Genes involved in ethylene biosynthesis (ACS2) [START_REF] Denoux | Activation of defense response pathways by OGs and Flg22 elicitors in Arabidopsis seedlings[END_REF], ethylene response (ERF1, LEA4-1) [START_REF] Lorenzo | ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense[END_REF] or to the response of abscisic acid (ABI1)(Leung et al, 1997) were not induced by latB. Therefore, it seems that latB action is not "universal", that it does not lead to a deregulation of the whole transcriptome. Its action seems to be directed mainly towards the SA signalling pathway or toward response to biotrophic pathogens.

Clearly, a SA dependent pathway is activated by latB. Expression of PR1, a classical representative of defence genes, is usually induced during pathogen attack, but also as a reaction to any SA increase. Besides, ICS1, that is responsible for most of the SA accumulation in response to bacteria [START_REF] Li | Signaling Crosstalk between Salicylic Acid and Ethylene/Jasmonate in Plant Defense: Do We Understand What They Are Whispering?[END_REF][START_REF] Wildermuth | Isochorismate synthase is required to synthesize salicylic acid for plant defence[END_REF][START_REF] Zheng X-Y | Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid[END_REF], was strongly induced. Therefore, the question was the following: did the changes in gene expression, that target SA responsive genes, occur through a SA-dependent pathway. We could show that the expression of PR1 and WRKY38 by latB relied on SA accumulation and a functional PAD4 protein. The fact that ICS1 expression is controlled by PAD4 in response to pathogen was established [START_REF] Cui | A core function of EDS1 with PAD4 is to protect the salicylic acid defense sector in Arabidopsis immunity[END_REF]).

Yet, the expression of PR2 and ICS1 is independent on SA, as seen in the NahGpi4kß1ß2. PR2 is one of the early defence genes, usually co-expressed with PR1 and PR5 and used as a marker of SA pathway [START_REF] Hamamouch | Expression of Arabidopsis pathogenesisrelated genes during nematode infection[END_REF]. Nevertheless, here we describe its clear SA-independent induction. It might seem contradictory, unless we take into account that PR2 protein is β-1,3glucanase, particularly important for antifungal defences [START_REF] Ali | Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance[END_REF]. PR2 is crucial for callose degradation and contributes to the SA increase; its overexpression enhances resistance both to fungal and bacterial pathogens [START_REF] Oide | A novel role of PR2 in abscisic acid (ABA) mediated, pathogen-induced callose deposition in Arabidopsis thaliana[END_REF].

It thus appears that latB (likely through its action on actin filaments) activates a signaling pathway typical of the response to pathogens. It activates PAD4. Some of the PAD4 action leads to ICS1 expression, to SA accumulation and to the expression of SA responsive genes. Yet, a part of PAD4 action is not dependent on SA, as seen by ICS1 expression. LatB also triggers a pathway that does not involve PAD4 nor SA, and that leads to callose accumulation and PR2 expression.

The action of latB is likely to occur through actin filament depolymerization. Actin filaments are involved in the control of vesicle trafficking. Phosphoinositides are also involved in vesicular trafficking. There is indeed an interaction/interplay between actin filaments and phosphoinositides [START_REF] Pleskot | When fat is not bad: the regulation of actin dynamics by phospholipid signaling molecules[END_REF]. F-actin capping protein interaction with actin monomers is regulated by phosphatidic acid and phosphatidylinositol-4,5-bisphosphate, a phosphoinositide [START_REF] Pleskot | Structural Insights into the Inhibition of Actin-Capping Protein by Interactions with Phosphatidic Acid and Phosphatidylinositol (4,5)-Bisphosphate[END_REF]. In pollen tube, cytoskeletal disruption by latB or jasplakinolide not only affects actin and arrested tube growth, but also causes relocalization and even dissociation of RabA4a and RabA4b-tagged vesicles [START_REF] Zhang | Interdependence of Endomembrane Trafficking and Actin Dynamics during Polarized Growth of Arabidopsis Pollen Tubes[END_REF]. Interestingly, PI4Kβ1 and PI4Kβ2 were shown to be recruited by RabA4b. Due to this interaction between actin and phosphoinositides in relation to vesicle trafficking, we were interested to investigate whether the action of latB could be impacted by alteration in phosphoinositide metabolism. To address this, pi4kß1ß2 mutant was a tool of choice. As already mentioned, PI4Ks are the first enzymes that commit PI toward the synthesis of phosphoinositides, and the beta isoforms PI4Kβ1 and PI4Kβ2 were shown to be recruited by RabA4b. PI4Kbeta proteins participate in cytokinesis in Arabidopsis roots by targeting actin filaments to the cell plate [START_REF] Lin | A dual role for cell plate-associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis[END_REF]. Nevertheless, it is important to consider the fact that pi4kß1ß2 plants exhibit constitutive activation of SA-related genes and high basal levels of callose. Yet it is true for mature 4 week-old soil-grown plants (Antignani et ST3), nor constitutively high expression of PR1 and ICS1 was observed (fig. 6).

The actin cytoskeleton of pi4kß1ß2 mutants appear to be more sensitive to latB (fig. 4). This is consistent with the above mentioned references which show F-actin capping protein interaction with actin monomers is regulated by phosphatidic acid and phosphatidylinositol-4,5bisphosphate, a phosphoinositide [START_REF] Pleskot | Structural Insights into the Inhibition of Actin-Capping Protein by Interactions with Phosphatidic Acid and Phosphatidylinositol (4,5)-Bisphosphate[END_REF]. In response to latB, pi4kß1ß2 seedlings accumulated callose, in a SA-and PAD4-independent way, as observed in WT plants. Yet, when we assay the SA increase in response to latB, a process not observed in WT is then detected: the SA induction in pi4kß1ß2 background still occurs, despite the mutation in PAD4 or expression of NahG. The pi4kß1ß2 double mutation allows to reveal a signalling pathway, triggered here by latB, that leads to SA biosynthesis and PR1 expression independently on PAD4. Yet, the PR1 expression in response to latB was not only observed in NahGpi4kß1ß2 and pad4pi4kß1ß2 triple mutants, but also in sid2pi4kß1ß2 (data not shown). Therefore, our data on the existence of such a SA independent pathway are strong. Is this pathway usually actively inhibited by functional PI4Ks? Is it linked to the effect of pi4kß1ß2 double mutation on trafficking? Or is it due to a role of phosphoinositide on signalling? An important contribution may come from immune-related MAPKs, whose transient or sustained activation works as a switch between pattern-triggered immunity and effector triggered immunity [START_REF] Tsuda | Dual regulation of gene expression mediated by extended MAPK activation and salicylic acid contributes to robust innate immunity in Arabidopsis thaliana[END_REF]. Indeed, Tsuda et al. described expression of PR1 in sid2 background, induced by constitutive activation of MPK3 and MPK6.

Another enzyme of the cascade, MPK4, was shown to interact with PI4Kβ1 during cell plate formation in cytokinesis [START_REF] Lin | A dual role for cell plate-associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis[END_REF]). More research is necessary to understand and characterize this pathway.

To summarize, we here report the defence-like consequences of actin cytoskeleton disruption by latB (fig. 7). Two kinds of signalling pathways were activated by latB treatment: a first pathway is independent on SA; it leads to the expression of genes such as PR2, but also to callose accumulation through PMR4. The second pathway involves SA synthesis via PAD4 and ICS1; it leads to the expression of genes such as PR1 and WRKY38. The double mutation in PI4Kß1ß2 makes the effect of latB on filaments more pronounced. Interestingly, the SA increase and PR1 expression in response to latB in the pi4kß1ß2 background is also triggered in an unconventional, PAD4-independent pathway. Either this pathway is actively inhibited in WT background by PI4Kß1ß2 (and thus not seen), or it is activated only when the PI4Kß1ß2 are missing. Is it the disrupted actin per se that act? Or are these pathways activated as a response to a process altered due to lack of good filaments? Such a process could be a modified vesicle trafficking due the missing actin filaments. In this regard, it would be interesting to monitor the processing of receptors of MAMPs in seedlings submitted to artificial cytoskeleton depolymerization agents.

We believe this work sheds light on the connection between cytoskeleton dynamics, vesicular trafficking and immunity at the cellular and subcellular level and thus will contribute to better understanding of plant responses to changing environment. PdF1.2a 1.03 ± 0.29 3.48 ± 0.64 2.22 ± 0.78 2.25 ± 0.32 1.84 ± 0.21 4.50 ± 0.70 1.72 ± 0.18 4.79 ± 0.92 3.49 ± 0.54 13.29 ± 2.34 4.67 ± 0.67 4.91 ± 0.96 PdF1.2c

Supplemental table ST1. Primers used in this study

Gene

1.17 ± 0.43 4.64 ± 1.05 1.82 ± 0.66 2.78 ± 0.37 2.15 ± 0.31 6.00 ± 1.05 1.73 ± 0.20 6.93 ± 1.55 2.34 ± 0.34 16.99 ± 5.05 4.15 ± 0.69 5.75 ± 1.25 ACS2

3.83 ± 2.15 2.20 ± 0.16 3.07 ± 0.33 4.97 ± 1.12 8.81 ± 1.30 42.83 ± 7.80 0.47 ± 0.18 2.13 ± 0.21 5.05 ± 0.87 10.43 ± 1.26 1.40 ± 0.12 10.30 ± 3.72 AOS 1.14 ± 0.39 6.07 ± 0.87 0.48 ± 0.15 1.27 ± 0.44 1.26 ± 0.67 2.05 ± 1.24 0.62 ± 0.14 2.15 ± 0.28 1.60 ± 0.76 5.08 ± 1.14 2.69 ± 0.92 5.82 ± 1.63 ERF1

2.05 ± 0.80 8.57 ± 0.55 1.09 ± 0.12 5.82 ± 0.55 2.86 ± 0.33 13.32 ± 1.90 0.82 ± 0.27 5.99 ± 0.68 2.51 ± 0.80 13.83 ± 0.92 3.13 ± 0.23 11.10 ± 1.91 LEA4-1

1.14 ± 0.32 1.35 ± 0.23 1.21 ± 0.24 0.87 ± 0.24 0.82 ± 0.15 1.29 ± 0.19 0.93 ± 0.12 0.97 ± 0.07 0.95 ± 0.22 1.25 ± 0.29 3.45 ± 2.07 0.92 ± 0.13 ABI1 0.98 ± 0.04 1.18 ± 0.03 0.89 ± 0.05 1.13 ± 0.06 0.00 ± 0.00 0.00 ± 0.00 0.56 ± 0.02 0.79 ± 0.03 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

pad4

  al, 2009; Thiele et al, 2009), pollen development (Záveská Drábková & Honys, 2017), cell-to-cell communication through plasmodesmata (Cui & Lee, 2016), and various stress responses. Arabidopsis genome encodes twelve callose synthases, each of a specific function (Chen & Kim, 2009). Callose synthase 12 (also known as GLUCAN-SYNTHASE LIKE 5, GSL5, of POWDERY MILDEW RESISTANT 4, PMR4) is the one responsive to wounding and pathogen responses (Ellinger et al, 2014; Keppler et al, 2018). Callose biosynthesis in the apoplast and its accumulation highly depend on the transport of the substrates outside of the cells. Notably, distinct signaling pathways may result in callose formation. For instance, wounding-induced and MAMP-triggered callose might represent different branches of responses going through PMR4

Figure 1 .

 1 Figure 1. Latrunculin B (latB) induces PMR4-dependent callose accumulation. Ten days old A. thaliana seedlings were treated for 24h with 0.05% DMSO (mock) of 200 nM latB. A. Representative images of aniline blue stained cotyledons of WT, pmr4, pUBQ::PMR4-GFP in pmr4, and pUBQ::PMR4-GFP in WT, epifluorescent microscopy, scale bar = 500µm. B. Quantification of the relative area occupied with callose particles, n=18. Values for the mocktreated plants are presented in white boxes, latB treated -in dark grey boxes. In the plots, central line represents the median occupancy, cross represents the mean, bottom and top edges of the box are 25 and 75% of distribution and the ends of whiskers are set at 1.5 times the interquartile range. Values outside this range are shown as outliers. Different letters indicate variants that are significantly different, one-way ANOVA with Tukey-HSD test, p<0.05. C. Relative expression of PMR4. Relative expression of PMR4 in WT plants. Values were normalized for the untreated WT.

Figure 2 .

 2 Figure 2. SA-independent induction of callose accumulation by latB treatment. Ten-day-old seedlings of WT, NahG, sid2 and pad4 were treated by 200 nM latB or 0.01 % DMSO as the control. Seedlings were harvested 24 hr after the treatment A. SA content. *** -samples are different, p<0.001, two-tailed t-test. B. Representative images of aniline blue stained cotyledons, scale bar 500 µm. C. Relative area of callose particles. Central line of the boxplot represents the median occupancy, cross represents the mean, bottom and top edges of the box are 25 and 75% of distribution and the ends of whiskers are set at 1.5 times the interquartile range. Values outside this range are shown as outliers. Different letters indicate variants that are significantly different, one-way ANOVA with Tukey-HSD test, p<0.05. At least 30 independent cotyledons were analysed for each variant.

Figure 3 .

 3 Figure 3. SA-independent expression of defence-related genes by latB treatment. Ten-dayold seedlings of WT, NahG and pad4 were treated by 200 nM latB or 0.01 % DMSO as the control. Seedlings were harvested 24 hr after the treatment. Data from the representative experiment are shown. Values were normalized for mock treated WT, note that data are plotted at the log10 scale. Asterisks indicate values, different from respective controls for each genotype, * -p<0,05; *** -p<0.001, two-tailed Student t-test). Reference gene TIP41.

Figure 4 .

 4 Figure 4. Effect of latB on actin filaments organization of pi4kß1ß2 mutants. A. Representative images of maximum intensity projections of 10µm abaxial epidermis of LifeAct-GFP expressing WT and pi4kß1ß2 plants. Confocal microscopy, scale bar = 20µm. Quantification of relative area occupied with fluorescent filament in WT (B) and pi4kß1ß2 (C). For each variant at least 15 independent seedlings were imaged, n=12. For quantification, maximµm intensity projections of 10µm Z-stacks of abaxial sides of the leaves were used. At least 30 regions of interest (ROI) measured for each treatment. In the plots, central line represents the median occupancy, cross represents the mean, bottom and top edges of the box are 25 and 75% of distribution and the ends of whiskers are set at 1.5 times the interquartile range. Values outside this range are shown as outliers. Asterisks indicate statistically significant differences with the mock treatment, Student ttest, p<0,05

Figure 5 .

 5 Figure 5. LatB induced SA and callose accumulation in pi4kß1ß2 mutants. Ten-days-old seedlings were treated by 200 nM latB or 0.01% DMSO as mock control. Seedlings were harvested 24 hr after the treatment. A. SA content. Data from two independent experiments are plotted. B. Relative area of callose particles in the cotyledons. At least 30 independent cotyledons were analysed for each variant. Experiment was repeated three times with similar results, the data from a representative repetition are shown. In boxplots, central line represents the median occupancy, cross represents the mean, bottom and top edges of the box are 25 and 75% of distribution and the ends of whiskers are set at 1.5 times the interquartile range. Values outside this range are shown as outliers. Different letters mark significantly different values, p<0,05; Student t-test, unequal variance.

Figure 6 .

 6 Figure 6. LatB-induced gene expression in pi4kß1ß2 mutants. Ten-days-old seedlings of WT, pi4kß1ß2, NahGpi4kß1ß2 and pad4pi4kß1ß2 were treated by 200 nM latB or 0.01% DMSO as mock control. Seedlings were harvested 24 hr after the treatment. Data are normalized to mocktreated WT, note that data are plotted at the log10 scale. Asterisks indicate values, different from

  al, 2015; Kalachova et al, 2019; Šašek et al, 2014). In younger plants, no PR1 overexpression was detected (Sasek et al., 2014). In the present study we used 10-day-old seedlings grown in vitro. In these conditions, neither SA accumulation (fig.5 A, Supplementary table

Figure 7 .

 7 Figure 7. Graphical summary of the latrunculin B effects in seedlings. The disruption of actin polymers by latB activates two kinds of signalling pathways. A first pathway is independent on SA; it leads to the expression of genes such as PR2, but also to callose accumulation. The second pathway involves SA synthesis via PAD4 and ICS1; it leads to the expression of genes such as PR1 and WRKY38. The double mutation in PI4K beta1 and beta2 makes the effect of latB on filaments more pronounced. Interestingly, the PR1 expression in response to latB is no longer SAdependent in the pi4kß1ß2 background: a SA independent pathway leading to PR1 expression is thus revealed. Still several open questions remain: what makes actin depolymerization trigger these pathways?

table ST2 .

 ST2 Effect of latB on defence gene expression.

	Supplemental
		Accession number	Forward primer	Reverse primer
	TIP41	AT4G34270	GTGAAAACTGTTGGAGAGAAGCAA	TCAACTGGATACCCTTTCGCA
	PR1	AT2G14610	AGTTGTTTGGAGAAAGTCAG	GTTCACATAATTCCCACGA
	PR2	AT3G57260	TATAGC CAC TGA CAC CAC	GCCAAGAAACCTATCACTG
	ICS1	At1g74710	GCAAGAATCATGTTCCTACC	AATTATCCTGCTGTTACGAG
	ICS2	AT1G18870	TGTCTTCAAAGTCTCCTCTG	CTTCCTCCAAACTCATCAAAC
	WRKY38	At5g22570	GCCCCTCCAAGAAAAGAAAG	CCTCCAAAGATACCCGTCGT
	LOX2	AT3G45140	ATCCCACCTCACTCATTACT	ATCCAACACGAACAATCTCT
	VSP2	AT5G24770	CCAAACAGTACCAATACGAC	CTTCTCTGTTCCGTATCCAT
	PAL1	AT2G37040	GTGTCGCACTTCAGAAGGAA	GGCTTGTTTCTTTCGTGCTT
	PAL2	AT3G53260	GTGCTACTTCTCACCGGAGA	TATTCCGGCGTTCAAAAATC
	PAL3	AT5G04230	CAACCAAACGCAACAGCA	CTCCAGGTGGCTCCCTTTTA
	PAD4	AT3G52430	GGTTCTGTTCGTCTGATGTT	GTTCCTCGGTGTTTTGAGTT
	BAP1	At3g61190	ATGCCCATCAATGGTAATG	TCCCACACTTATCACCAAA
	PDF1.2a	AT5G44420	ACGCACCGGCAATGGTGGAA	TGCATGATCCATGTTTGGCT
	PDF1.2c	AT5G44430	CTGCTACCATCATCACCTTC	CCGCAAACGCCTGACCATGT
	AOS	AT5G42650	GGTCATCAAGTTCATAACCG	TTTCTCAATCGCTCCCAT
	ACS2	At1g01480	GTTAAGCTCAATGTGTCTCC	AAGCAAATCCTAAACCATCC
	ERF1	At3g23240	ATCAAATCCGTAAGCTCAAG	CCAAACCCTAATACCGTTTC
	LEA4-1	AT1G32560	AGCAAAAGCTGATGAGAAGGCA	TAGGTCTGAGGAGGCACTGA
	ABI1	AT4G26080	ATGTCGAGATCCATTGGCGAT	TTCCTTTCTCCGCTCATCCG
	PMR4	AT4G03550	ATGTACACATGTAAATGGCG	GAGGCTGGGTCTCGAGGAGTT

Supplemental table ST3. Effect of latB on salicylic acid content in seedlings

  . Seedlings were grown in vitro for 10 days and treated with 1µM latB for 24h. DMSO was used as mock treatment. SA content was measured by LC-MS. Means ± SE from 8 values generated in two independent experiments are shown.

	WT		NahG				pi4k β1β2		NahGpi4kβ1β2	pad4pi4kβ1β2
	mock	LatB	mock	LatB	mock	LatB	mock	LatB	mock	LatB	mock	LatB
	SA content (ng/ g FW)
			DMSO		LatB
	WT		307,70 ± 66,59	7247,39 ± 793,85
	pmr4		402,10 ± 45,02 10179,89 ± 756,35
	NahG		64,11 ± 6,30	451,38 ± 51,03
	sid2		54,30 ± 6,84		54,84 ±	3,32
	pad4		102,97 ± 20,15	788,84 ± 158,57
	pi4k β1β2		159,56 ± 26,04	6879,74 ± 864,17
	NahGpi4kβ1β2 105,50 ± 18,14	2286,92 ± 222,89
	sid2pi4kβ1β2		60,94 ± 9,76		62,75 ± 10,80
	pad4pi4kβ1β2	237,77 ± 92,15	1925,06 ± 385,01

ACKNOWLEDGEMENTS

We are very grateful to Dr. Ivan Kulich for providing pUBQ::PMR4-GFP expressing lines and Dr.

Yvon Jaillais for mCitrine-2xFAPP1 expressing line. We would like to thank Dr. Kateřina Malínská for training and the precious advices with microscopy.

FUNDING INFORMATION

This work was supported by Czech Science Foundation [GAČR grant no. 17-05151S]. TK benefited from the Program of Postdoctoral Fellowships of the Czech Academy of Sciences [PPPLZ grant no. TK 919220]. OI received the Visegrad scholarship (2018-2019) [grant no. 51810647]. HL benefited from Charles University student grant [GAUK no. 992416]. The work was also supported by the Ministry of Education, Youth and Sports of CR from European Regional Development Fund-Project "Centre for Experimental Plant Biology" [grant no. CZ.02.1.01/0.0/0.0/16_019/0000738]. IEB imaging facility is supported by OPPK CZ.2.16/3.1.00/21519.


G Ounoughene

C Chivas-Joly

C Longuet

O Le Bihan

J-M Lopez-Cuesta

L Le Coq

Evaluation of nanosilica emission in polydimethylsiloxane composite during incineration

Keywords:

At the end of their life cycle, it is expected that many industrial silicone components end up in incineration waste plants. Hence, the issue concerning the risks resulting from the generation of fumes (combustion gas and aerosol) has to be addressed. The aim of our work was to investigate the behavior and fate of nanosilicas from filled polydimethylsiloxane nanocomposites burnt under two different scenarios of incineration. Combustion tests have been performed at lab-scale using a particular tubular furnace and a specific cone calorimeter. The collected fumes (particulate matter and gas phase) have been characterized using various techniques. The results show persistent nanosilica particles, newly produced nanosilica particles in the fumes and in the residues, as well as silicon oxycarbide SixOyCz particles which seem to originate from polysiloxane matrix decomposition.

Introduction

Nanocomposites have been widely studied and produced since the end of the nineties. These trendy materials containing nanomaterials represent approximately 20,000 metric tons per year [START_REF] Keller | Predicted releases of engineered nanomaterials: from global to regional to local[END_REF]. This waste should be streamed to be recycled, placed in landfills or incinerated. Recently, few studies on the behavior and the fate of nano-objects in incineration facilities have been investigated at lab-scale [START_REF] Le Bihan | Characterization and reduction of particulate emissions from the incineration of wastes containing engineered NMs[END_REF] and at real scale [START_REF] Walser | Persistence of engineered nanoparticles in a municipal solid-waste incineration plant[END_REF].Various polymers have been studied, from paint where Massari et al. [START_REF] Massari | Behavior of TiO 2 nanoparticles during incineration of solid paint waste: a lab-scale test[END_REF] suggest that TiO 2 nanoparticles may undergo to physicochemical transformation during the incineration, and that titanium found in ashes stay in glass matrix. According to Derrough et al. [START_REF] Derrough | Behaviour of nanoparticles during high temperature treatment (Incineration type)[END_REF], the most of the nanomaterials remained in the bottom ash and only a small amount partitioned into the particulate matter. At the same time, Vejerano et al. [START_REF] Vejerano | Characterisation of particle emissions and fate of nanomaterials during incineration[END_REF] [7],) showed that the combination of high specific surface area of nanomaterials might be responsible for affecting the formation of toxic pollutants during incineration. In parallel, Ounoughene et al. [START_REF] Ounoughene | Behavior and fate of halloysite nanotubes (HNTs) when incinerating PA6/HNTs nanocomposite[END_REF] investigated the behavior of various nanocomposites during incineration process and show that nanoparticles (NP) should be potentially transformed into other structures which were found in both the aerosol and the residues. The risks related to the potential toxicity of fine particles (e.g. during combustion processes) have already been outlined [START_REF] Muller | Modeling the flows of engineered nanomaterials during waste handling[END_REF]. The review of the bibliography related to the incineration of filled polymer highlights the lack of studies concerning silicone incineration (excepted, a recent paper on characterization of waste containing nanofillers [START_REF] Tran | Characterization of polymer wate containing nano-fillers prior its end-of-life treatment[END_REF]. They focus only on solid form and don't present an overview of the fate of nanoparticles. The originality of this study lies in the fact that silicones are crosslinked materials and not recyclable and the only issues remains incineration or landfilling [START_REF] Laubie | Methodology to assess silicone (bio)degradation and its effects on microbial diversity[END_REF] and that the thermal degradation of silicone induces silica formation. The limited presence of incineration behavior of nanosilica filled polydimethylsiloxane in published literature has prompted this investigation. These widely used polymers have many industrial and domestic applications such as household and personal care products, textile treatments, electrical cables and wires [START_REF] Colas | Silicones: preparation, properties and performance[END_REF][START_REF] Horii | Survey of organosilicone compounds, including cyclic and linear siloxanes, in personal-care and household products[END_REF]. As a consequence of their widespread use, many end-of-life polysiloxanes components are likely to be sent to municipal waste incineration plants. Polysiloxanes can enter a waste incineration plant in two different ways: (1) solid waste silicones are converted back to inorganic substances, mainly, amorphous silica and carbon dioxide (polysiloxanes can also be landfilled), (2) liquid waste from sludge (resulting from products such as shampoos or detergents) which ends up in municipal wastewater [START_REF] Colas | Silicones: preparation, properties and performance[END_REF]. However, only few studies focus on the silica produced during the combustion of polysiloxanes. In biogas industries, problems have been reported regarding silica particles generated during the combustion of biogas containing siloxane traces. Indeed, the deposition of these particles in the combustion chamber causes dysfunction of engines and turbines [START_REF] Turkin | Deposition of SiO 2 nanoparticles in heat exchanger during combustion of biogas[END_REF][START_REF] Tansel | Oxidation of siloxanes during biogas combustion and nanotoxicity of Si-based particles released to the atmosphere[END_REF][START_REF] Dewil | Energy use of biogas hampered by the presence of siloxanes[END_REF]. To our knowledge, apart from this last issue, studies dealing with silica particles (mainly submicron particles) released from combustion of polysiloxanes are scarce. Hence, further investigations about fumes (combustion gas and aerosol) produced during the combustion of polydimethylsiloxanes are required. Polydimethylsiloxane (PDMS) is the main commercial polysiloxane. It is a synthetic polymer with SieO backbone and methyl side groups. It exhibits interesting properties like hydrophobic characteristics [START_REF] Owen | Siloxane surface-activity[END_REF], superior properties of heat and cold resistance, excellent weather resistance and electrical properties [START_REF] Hayashida | Silicone rubber composition having excellent heat resistance[END_REF]. Thermal degradation of PDMS leads to a silica residue or a silicon oxycarbide residue depending on combustion conditions [START_REF] Hamdani | Flame retardancy of silicone-based materials[END_REF][START_REF] Camino | Thermal polydimethylsiloxane degradation. Part 2. The degradation mechanisms[END_REF][START_REF] Camino | Polydimethylsiloxane thermal degradation Part 1. Kinetic aspects[END_REF]. Furthermore, in order to improve their mechanical properties, PDMS are often reinforced with nanoparticles like amorphous silica [START_REF] Hamdani | Flame retardancy of silicone-based materials[END_REF][START_REF] Industries | Basic Characteristics and Applications of AEROSIL® products[END_REF] which can be divided into manufactured products and natural minerals (like diatomaceous earth). Synthetic amorphous silica (SAS), especially nanosized silica (with a diameter < 100 nm), has received huge attention towards many industrial applications for several years. Even if, many aspects related to the size and porous texture of these nanomaterials have raised in the literature. That concerns mainly safety since human exposure to SAS and environmental release [START_REF] Napierska | The nanosilica hazard: another variable entity[END_REF][START_REF] Polloth | The toxicological mode of action and the safety of synthetic amorphous silica-a nanostructured material[END_REF]. However, papers regarding risks assessment in link with the incineration end-of life deals rarely with the impact of particles size after thermal degradations.

The thermal degradation of nanosilica filled PDMS was studied by different authors [START_REF] Hamdani | Flame retardancy of silicone-based materials[END_REF][START_REF] Camino | Polydimethylsiloxane thermal degradation Part 1. Kinetic aspects[END_REF][START_REF] Kashiwagi | Flame retardant mechanism of silica -effects of molecular weight of resin[END_REF] and it was shown that nanosilica particles decrease the flammability of PDMS. However, the fumes produced during the thermal degradation were never investigated, and tests involving the combustion parameters of domestic waste incineration (temperature around 850 °C, highly ventilated combustion, at least 2 s residence time for the combustion gas in a post-combustion chamber at 850 °C, and high oxygen/fuel contact) were never reported. Here again, there is a lack in the literature regarding silica particles behavior during combustion, especially during incineration.

For the first time, the present study brings an insight about silica particles generated by the combustion (under two incineration scenarios) of a silica/PDMS nanocomposite. The authors suggest the use of two small scale combustion devices with controlled key incineration parameters: a modified tubular furnace and a modified cone calorimeter to access of PN (particle number) by combining physical one-line characterization [START_REF] Ounoughene | Behavior and fate of halloysite nanotubes (HNTs) when incinerating PA6/HNTs nanocomposite[END_REF] and particles sampling (morphological analysis). The fumes were sampled downstream of the combustion devices (downstream of the post-combustion chamber is considered as equivalent to the upstream of fumes treatment sections in a real-scale incineration plant), and were characterized using various aerosol and gas analysis techniques. The solid combustion residues were analyzed as well. The aim of this work was (i) to study the behavior and fate of silica particles when a silica/PDMS nanocomposite is incinerated, (ii) to simulate and to compare the results of two experimental setups devoted to incineration at lab scale (tubular furnace and cone calorimeter with controlled atmosphere). This study also investigated possible modifications affecting nano-silica particles undergo during their stay in the incinerator furnace chambers, and where they could be potentially released.

Methodology

Two experimental setups were used in order to carry out incineration tests and to track effluents (particles and smoke). Two lab-scale devices (tubular furnace and cone calorimeter) were improved and adapted to simulate incineration, in accordance with the 4 T (Temperature, Time, Total amount of O 2 and Turbulance) rules [START_REF] Ounoughène | Etude des emissions liées à la decomposition thermique de nanocomposites: Application à l'incinération[END_REF]:

-Temperature (850 °C) -Time (2 s) -Total amount of Oxygen (> 11%) -Turbulence (a good mix between combustible and oxygen) This section aims to compare these two devices to ensure the key incineration parameters are under controlled.

Sample's description and formulations

The sample is a nanosilica filled polydimethylsiloxane. It consists of a PDMS gum (68.5 wt%), a siloxane oil (3 wt%, PDMS-SiOH) and nanosized fumed amorphous silica particles (27 wt%) with an average primary particle size of 12 nm. PDMS was mixed with a 2,4 dichlorobenzoic catalyst (used as radical generator necessary to cross linked the matrix) and thermo-compressed using a Darragon type 3 press in 10 × 10 × 0.4 cm 3 steel square (at 30 sheets, 115 °C during 8 min). A pristine PDMS is also tested for comparison, when needed. 0.25 g samples (1 × 1 × 0.2 cm 3 ) from these specimens were cut and then tested in the incineration scenario 1 (tubular furnace) and 2.5 g (2.5 × 2.5 × 0.4 cm 3 ) were tested in the incineration scenario 2 (cone calorimeter).

Lab-scale incinerator devices and analyses

A tubular horizontal furnace (Carbolite STF 15/610) [START_REF] Le Bihan | Characterization and reduction of particulate emissions from the incineration of wastes containing engineered NMs[END_REF] and a cone calorimeter (Fire Testing Technology) [START_REF] Ounoughène | Etude des emissions liées à la decomposition thermique de nanocomposites: Application à l'incinération[END_REF] were modified to simulate conditions implemented in an industrial grate incinerator. As illustrated in Fig. 1, the sample holder is considered as the "combustion chamber" where the temperature is maintained at 850 °C (at least) for the tubular furnace and where the thermal flow is settled at 75 kW/m² (which corresponds to a temperature close to 850 °C) in the cone calorimeter. Air cross flows penetrate and mix with the combustibles. Then, the "post-combustion chamber" is the chamber zone where the fumes continue their oxidation (during 2 s for the tubular furnace; during 0.2 s for the cone calorimeter). Finally, it is conveyed to the measurement lines described as follows.

In order to superimpose the curves of time tracking and to synchronize all events detected, it is important to know the response time of each measurement line. It was determined by stopwatch (an uncertainty of two seconds was taken into account).

The fumes were sampled appropriately as explained below and analyzed in two different ways:

-time tracking for the gas and particles concentration from the fumes, -off-line analysis for the morphology and chemical composition of particles.

Further analyses were conducted on the combustion solid residues (see below, Section 2.2.4).

Time tracking for the gas phase

The sampling of the gas phase from the fumes followed the methods used for sampling direct emissions from stationary combustion sources. For both devices, the fumes were dried and filtered using a line with a hot filter and a condenser. Multi-gas analyzers were used (PG-250 Horiba portable gas analyzer for the tubular furnace and Servomex 4100 gas analyzer for the cone calorimeter) to determine the evolution of the combustion gases concentrations: consumption of O 2 (% vol. ), production of CO 2 (% vol. ) and CO (ppm vol. ).

Time tracking for particle number concentration

The fumes sampled have to be representative of the aerosol upstream the flue-gas cleaning systems, i.e. downstream of the postcombustion chamber. For the two devices, the target temperature is 150 ( ± 10) °C. Actually, for industrial conditions, the flue gas leaves the post-combustion chamber and goes through a heater (for heat recovery purpose) and then flows through a treatment process at a temperature around 150 ( ± 10) °C in accordance with BREF Waste Incineration (2006) [START_REF]Reference Document on Best Available Techniques for the Waste Treatments Industries[END_REF]. For each scenario, two different samples and counters were used to conduct the analysis with respect to the temperature constraint mentioned above.

2.2.2.1.

Incineration scenario 1: tubular furnace device. Regarding the tubular furnace scenario, an Electrical Low Pressure Impactor (ELPI, Dekati) was used downstream of a FPS dilutor (Fine particle sampler, Dekati). As explained in a previous study [START_REF] Hamdani | Flame retardancy of silicone-based materials[END_REF], the objective of the sampling was to avoid cold spots below 150 ( ± 10) °C during the first dilution and then thermophoretic losses can be prevented by heating the sampling probe to same or higher temperature than sample temperature. The FPS dilutor performed two successive dilutions: the first heated dilution at 150 ( ± 10) °C and the second at room temperature. Thus, the sampled fumes underwent two dilutions with a total factor of 1:30. The ELPI provided a real-time measurement of particle number concentration through 12 channels from 17 nm to 5 μm (aerodynamic diameter of sphere-like particles) [START_REF] Keskinen | Electrical low pressure impactor[END_REF].

2.2.2.2. Incineration scenario 2: cone calorimeter device. Regarding the cone calorimeter device, an APS spectrometer (TSI, Aerodynamic Particle Sizer 3321) and a DMS 500 spectrometer (Cambustion M85, Fast particle analyser, Dynamic particle spectrum) were used downstream of two dilution stages: a first hot dilution with a DI1000 dilutor (Dekati) with a 1:10 dilution ratio followed by two VKL10 dilutors (Palas) with a 1:100 dilution ratio. Similarly to the scenario 1, the first dilution was heated at 150 ( ± 10) °C in order to avoid cold spots and the second dilution was at room temperature. Thus, the sampled fumes underwent three dilutions with a total theoretical factor of 1:1000. The DMS500 and the APS provided a real-time measurement of particle number concentration respectively from 5 nm to 1 μm (electrical mobility diameter of sphere-like particles) and from 1 to 2 μm (aerodynamic diameter of sphere-like particles) [START_REF] Biskos | Description and theoretical analysis of a differential mobility spectrometer[END_REF][START_REF] Symonds | Diesel soot mass calculation in real-time with a differential mobility spectrometer[END_REF][START_REF] De Carlo | Particle morphology and density characterization by combined mobility and aerodynamic diameter measurements, part 1: theory[END_REF].

The PN x-y are defined as the number concentration of particles counted by the ELPI, the APS and the DMS500 with mean geometric diameter (Di) between x and y μm.

A previous study [START_REF] Chivas-Joly | Characterization of aerosols and fibers emitted from composite materials combustion[END_REF] have shown that the three counters from the measurement lines indicate different diameters (aerodynamic diameters for ELPI and APS, electrical mobility diameters for DMS) since they use different techniques to count particles. So, it must be borne in mind that the diameters are not equivalent.

Off-line analysis for particulate matter

Regarding off-line analyses, fumes and particles were collected over a targeted time range on a TEM grid with a MPS ® (Mini-Particle-Sampler, Ecomesure [START_REF] R'mili | Particle sampling by TEM grid filtration[END_REF]). It was a 10 s sampling downstream of the tubular furnace and downstream of the first dilution stage for the cone calorimeter, as presented on Fig. 1. A Philips CM12 TEM 200 kV was used for the imagery.

Analysis of the combustion residues

The combustion solid residues contained in the sample holder of the lab-scale incinerators were collected after each test. They were analyzed by scanning electronic microscope (SEM, JEOL 7600F High Resolution Analytical SEM energy dispersive spectrometer SDD BRUKER (EDS) X-ray detector), by X-Ray Diffraction (XRD) with a Bruker AXS D8 Advance diffractometer using Cu Kα radiation, and by a Bruker Optics IFS 66 series FT-IR spectrometer.

Standard cone calorimeter test

A Cone Calorimeter from FTT (United Kingdom), according to the standard ISO 5660, has been used in order to measure the HRR (heat rate release) and the mass loss during the tests. The heat flux was fixed to 75 kW/m² and sample size to 100 × 100 × 4 mm. Ignition was piloted using a spark igniter. At least two samples were tested for each formulation and reproducibility was considered as satisfactory. The uncertainty on main values is considered around 15%.

Results and discussion

Incineration experiments were performed through both scenarios.

The time tracking results gave information on the thermal decomposition of the nanocomposite and imagery techniques on the morphology of the fumes particles. Besides, the analysis of residues brought supplementary information on the fate and the behavior of silica particles.

Time tracking of gas concentration and particle number concentration

The graphs showing the evolution of concentrations of different gases during the two incineration scenarios of the nanocomposite are given in Fig. 2. The averages of three runs are presented with the associated standard deviation.

The first observation is a rather good agreement of time tracking results for the two incineration scenarios. Despite the differences in the amount of samples tested in the combustion devices (0.25 g for tubular furnace versus 2.5 g for cone calorimeter) and in the setups of measurement lines used, close tendencies were observed. Indeed, regarding the gas time tracking, the dioxygen (O 2 ) consumption is symmetrical to the carbon dioxide (CO 2 ) production for both cases. Also, the production of carbon monoxide (CO) follows a two-step mechanism with a first slight emission followed by a larger emission when the CO 2 is decreasing, in both cases.

Besides, the flame appears at t = 30 s and disappears at t = 125 s, in the case of the cone calorimeter scenario (for which the flame can be observed through a window). Then, according to the Fig. 2b, the two step mechanism mentioned above can be related to the ignition phenomenon. Actually, when the flame appears, the temperature in the post-combustion chamber increases and reaches around 1000 °C (according to the thermocouple situated in the post-combustion chamber between the surface of the sample and the base of the cone calorimeter). During this elevation of temperature, O 2 is consumed, CO 2 and particles (PN 0,1 and PN 0,1-1 ) are emitted significantly while CO is very slightly produced. Thus, this first phase could represent a complete combustion whereas the second phase could represent an incomplete combustion during which CO is highly emitted, little CO 2 is produced and hardly any O 2 is consumed. The temperature also decreases during this phase. Furthermore, this behavior can be explained by the decomposition pathway of PDMS material. Actually, this nanocomposite can be considered as an "intumescent" material because it swells when it is submitted to a heat source and because a silica protective layer appears on its surface [START_REF] Hamdani | Flame retardancy of silicone-based materials[END_REF]. Thus, the silica layer acts as a barrier limiting mass and heat transfers. In this way, the two step mechanism observed could be related to the silica layer evolution during the combustion. After the ignition, the progressive swelling of the material and the formation of a white layer on the surface with a complete combustion can be observed. Then, after the extinction, this layer protects the remaining combustible material from oxygen. In this way, the oxidation cannot be completed. This would explain why CO predominates over CO 2 during this second step.

Regarding the particles time tracking, for PN 0,1 (i.e particles with diameter < 100 nm), for both scenarios, they are released even before the ignition, due to pyrolysis of PDMS and de-cohesion of silica particles from the polymer matrix during the degradation. They are predominant after the beginning of combustion because of the formation of the barrier layer and the swelling of the material. Then, they decrease in favor of PN 0,1-1 (i.e. particles with diameter 100 nm < Di < 1 μm). During the combustion stage (Fig. 2b), the production of both categories of particles is quasi-constant. As for PN 1-2 (i.e. particles with diameter 1 μm < Di < 2 μm), these particles appear later in the combustion process and they are relatively less abundant. It can be explained by the temperature reached during the flame (around 1100 °C), which is able to initiate sintering of silica particles [START_REF] Buscarino | Sintering process of amorphous SiO2 nanoparticles investigated by AFM, IR and Raman techniques[END_REF].

Additionally, it can be observed that the concentration of particles released is high and that agglomeration of these particles in the furnace is highly likely. However, it would be difficult to ensure if these phenomena occur in a real scale industrial furnace, given the size limits of the lab scale devices. Furthermore, the noticeable PN 0,1 trail in the end of the combustion, for both cases (but more accentuated for the second scenario with the cone calorimeter) can be explained on the one hand by a thermophoresis phenomenon in the measurement line; and on the other hand by the glowing of the solid residue (described hereinafter) which would be dragged by the air flow.

Imaging and X-ray microanalysis

The results from image analysis provide only qualitative considerations.

Fumes particles

For both scenarios, imaging on combustion particles collected on TEM grids reveals the presence of submicronic aggregates and agglomerates. The primary particles of these structures exhibit different sizes: nanoparticles (D < 100 nm) and ultrafine particles (D > 100 nm) (Figs. 3 and4).

The terms primary particles, aggregates and agglomerates are defined as follows: primary particles are the smallest identifiable individual particles, aggregates are primary particles contacting each other at surfaces or edges and they cannot be broken down, while agglomerates are particles witch can be severed or aggregates contacting each other but severable [START_REF] Industries | Basic Characteristics and Applications of AEROSIL® products[END_REF]35,[START_REF] Walter | Primary particlesagglomeratesaggregates[END_REF].

• Regarding aggregated nanoparticles, their diameter varies from 15 nm for the smallest particles to 50 nm for the largest. Their structures are chain-like and strung together like a fractal pearl. The size of aggregates varies from 100 to 500 nm. It should be noted that the silica used as fillers in PDMS has a bimodal population (around 10 nm and 40 nm).

• Regarding ultrafine particles, the primary particles are roughly spherical and their structures of about 1 μm size are also chain-like but the network seems more compact. X-ray microanalysis shows the Si/C/O ratios of these particles (Fig. 5). The finest primary nanoparticles ̴ 15 nm contains mainly silicon and oxygen, which corresponds to silica composition, despite the Si/O ratio is not accurate. The fine primary particles (> 100 nm) and the biggest nanoparticles (50 < D < 100 nm) contain Si, C and O elements. According to the literature, these compounds with this type of composition should correspond, partially, to silicon oxycarbides (Si x O y C z ) [START_REF] Camino | Thermal polydimethylsiloxane degradation. Part 2. The degradation mechanisms[END_REF][START_REF] Camino | Polydimethylsiloxane thermal degradation Part 1. Kinetic aspects[END_REF][START_REF] Bahloul-Hourlier | Reaction pathways during the thermal conversion of polysiloxane precursors into oxycarbide ceramics[END_REF].

Solid combustion residues

The Fig. 6 shows the meringue-like residue resulting from the combustion in the cone calorimeter (scenario 2). It reveals an "intumescent" behavior of the material which has swollen during the thermal decomposition. The same meringue-like residue is observed for scenario 1 as well.

Heat Release rate (HRR) and mass loss values of the silica filled PDMS as well as these of a pristine PDMS were registered and are presented Fig. 7a andb. It can be noticed that the presence of silica does not affect the value of the HRR peak, nevertheless the combustion is shorter in presence of silica. Consequently, the presence of silica seems to change the PDMS decomposition, despite the formation of a charred structure. PDMS degradation induced release of cyclo-siloxane gases in addition to silica. These gases promote expansion of the structure and also the generation of an intumescent layer.

Similarly, the final residue is obtained at a lower time for the filled PDMS. Moreover, the difference in the weight residue is lower (20%) than the initial loading of silica in the filled PDMS (27%) (Fig. 7b). Actually, pristine PDMS presents 40 wt.% residue at the end of the test and filled PDMS 60 wt.% in the same conditions. It can be suggested that a part of the silica initially incorporated is released in the aerosol or the presence of silica changes the degradation mechanism of PDMS and decrease the formation of silica.

SEM (coupled with X-ray microanalysis) performed on combustion residues reveals that the residues seem to consist of aggregates and agglomerates with three sizes of primary particles (Fig. 8): the finest nanoparticles (D < 15 nm), the intermediate nanoparticles (50 < D < 100 nm) and the biggest ultrafine particles (D > 100 nm).

XRD performed on solid residues (from both incineration scenarios) showed that the residue is amorphous.

FTIR performed on solid residues (from both incineration scenarios) showed SieOeSi bond (1033 cm -1 ) and SieOeC bond (813 cm -1 ) [START_REF] Camino | Thermal polydimethylsiloxane degradation. Part 2. The degradation mechanisms[END_REF] (Supporting information).

According to FTIR analysis and semi-quantitative X-ray microanalysis, this expanded residue from PDMS/27SiO 2 decomposition consists of silica and silicon oxycarbide (Si x O y C z ).

The origin of particles

The origin of the particles observed in residues and in aerosol was scrutinized. Actually, since the tested nanocomposite consists of PDMS and nanosilica particles (about 12 nm), the observed particles could come from the PDMS thermal decomposition or from the initially incorporated nanosilica particles. The determination of the origin seems difficult because the PDMS can produce silica and silicon oxycarbide via bonds reorganization under high heat rate, as shown in the literature [START_REF] Hamdani | Flame retardancy of silicone-based materials[END_REF][START_REF] Camino | Thermal polydimethylsiloxane degradation. Part 2. The degradation mechanisms[END_REF][START_REF] Camino | Polydimethylsiloxane thermal degradation Part 1. Kinetic aspects[END_REF]. Then, in order to attempt to assign the origin of each type of particles, the particles generated during the combustion of a virgin PDMS (pristine, without nanosilica) were observed (Fig. 9). The residues consist of aggregated/agglomerated fine particles (primary fine particles > 100 nm). The aerosol contains aggregated/agglomerated fine particles and nanoparticles: the finest primary nanoparticles around 15 nm, the intermediate primary particles between 50 and 100 nm, and the biggest primary particles > 100 nm). The X-ray microanalysis shows the presence of Si, C and O, but only median and bigger silica particles contain carbon. Hence, this suggests that these last kinds of silica result mainly from the decomposition of PDMS.

Both for filled PDMS and pristine PDMS, three sizes of particles are observed in the aerosol: the finest D ̴ 15 nm, the intermediate 50 < D < 100 nm, and the biggest particles > 100 nm.

The finest primary nanoparticles (15 nm) could come from the initially incorporated nanosilica but also from the thermal decomposition of PDMS, in the case of the filled PDMS. Emission of smallest primary nanoparticles is difficult to be determined unambiguously. The other particles may come from the decomposition of the polydimethylsiloxane matrix and/or from the re-organization via agglomeration/aggregation and possibly sintering of newly produced particles and initially present nanoparticles.

Conclusion

By means of two different experimental set-ups, it has been shown that the incineration of a nanosilica filled polydimethylsiloxane releases silica nanoparticles. Indeed, regarding its melting point (around 1700 °C [START_REF] Schweigert | Structure and properties of silica nanoclusters at high temperature[END_REF]), silica cannot be destroyed after an incineration process and it remains intact.

This study highlights that silica is a persistent product of the incineration of a nanosilica filled polydimethylsiloxane. Thus, it can enter the environment through incineration outflows and can raise concerns during slags and fumes treatments as well.

Therefore, it seems relevant to determine how silica nanoparticles could act on health and ecosystem.

Even if amorphous silica is not involved in progressive fibrosis of the lung and it is less dangerous than the crystalline silica (which is known for its inflammogenic, fibrogenic and carcinogenic activities [START_REF] Napierska | The nanosilica hazard: another variable entity[END_REF]), at high doses amorphous silica may result in acute pulmonary inflammatory responses.

Furthermore the nanosize of the silica makes it a potential danger. Many aspects related to the size of these nanomaterials have raised concerns about safety. As primary silica particles are generally nonisolated, some researchers consider that amorphous nanosilica particles are not harmful [START_REF] Polloth | The toxicological mode of action and the safety of synthetic amorphous silica-a nanostructured material[END_REF][START_REF] Dekkers | Presence and risks of nanosilica in food products[END_REF]. Besides, other researchers assert that even if nanosilica particles do aggregate, their specific area is the key parameter of their potential toxicity [START_REF] Napierska | The nanosilica hazard: another variable entity[END_REF][START_REF] Reijnders | The release of TiO 2 and SiO 2 nanoparticles from nanocomposites[END_REF]. Indeed, their surface can interact with the biological environment, so in this way, the size and surface physico-chemical features of these nanoparticles contribute decisively to their biological effects Thus, more detailed toxicity studies are required in order to supplement the existing data and to clearly identify the nanosilica health hazards.

The time tracking results revealed the barrier effect of silica particles in the polydimethylsiloxane matrix for both scenarios through a two-step mechanism. Despite clear differences in the experimental setup: in the measurement line as well as in the combustion chambers (i.e., the way the sample is heated and the residence time of combustion gases), we obtain convergent results which indicates that the filled PDMS decomposes in the same way regardless of the incineration scenario.

In addition, the results showed a persistence of the incorporated nanosilica particles and a production of nanosilica and silicon oxycarbide (SixOyCz) in the fumes and in the solid residues. The primary particles are aggregated/agglomerated and they present three different sizes: around 15 nm, between 50 and 100 nm, and > 100 nm. The finest particles could result, at least partially, from the silica introduced initially in the PDMS and from the PDMS decomposition as well. The biggest particles seem to come from to PDMS decomposition, due to the presence of carbon shown by X-microanalysis. The origin of the middle size particles, 50 nm < D < 100 nm, is not clearly identified. These particles could originate either from filled silica or from the PDMS decomposition, as well as a combination of both. The substitution of silica with isotropic silica can provide an interesting study for the future.

Fig. 1 .

 1 Fig. 1. Basic diagram of the two experimental setups (incinerator devices with measurement lines).

Fig. 2 .

 2 Fig. 2. Time tracking of gas concentration and particle number concentration from filled PDMS (a) Incineration Scenario 1: Tubular furnace -(b) Incineration Scenario 2: Cone calorimeter.

Fig. 3 .

 3 Fig. 3. Particles collected on TEM grids from filled PDMS (a) Incineration Scenario 1 (tubular furnace, t = [30,40]s) and (b) Incineration Scenario 2 (cone calorimeter, t = [50,60]s).

Fig. 4 .

 4 Fig. 4. Example of aggregates/agglomerates from fumes issued of filled PDMS -3 sizes of silica primary particles.

Fig. 5 .

 5 Fig. 5. X-ray microanalysis on fumes particulate matter from filled PDMS.

Fig. 6 .

 6 Fig. 6. Picture of the meringue-like solid residue of filled PDMS from initial sample of 100 × 100 × 4 mm.

Fig. 7 .

 7 Fig. 7. Standard Cone Calorimeter test. a) HRR measurement vs timeb) Mass lost during the tests.

Fig. 8 .

 8 Fig. 8. SEM on residues from filled PDMS. (a) Incineration scenario 1 -(b) Incineration scenario 2.

Fig. 9 .

 9 Fig. 9. Particles from pristine PDMS incineration in the cone calorimeter (scenario 2). (a) Combustion residues particles by SEM -(b) Combustion aerosol particles by TEM.

Acknowledgements

Financial support for this work was provided by ADEME, INERIS and LNE (Grant number 1106C0026).

The authors are grateful to their colleagues from INERIS, LNE, Trédi (Séché Environnement), C2MA (Centre des Matériaux des Mines d'Alès) and GEPEA (Mines Nantes), for their advice and technical support. Special thanks go to N. Stephant and O. Aguerre-Chariol for their respective contributions to SEM and TEM imaging.

Conflict of interest

The authors declare that they have no conflict of interest.