**2. Results**

#### *2.1. Non-Biological ROS Production by CaPPs*

We made the hypothesis that CaPPs could have a NP-like effects and could thus generate ROS independently of living cells. According to this hypothesis, we checked if CaPPs could induce ROS generation independently of any living cells. We showed by using the Murashige and Skoog (MS) culture medium that CaPPs spontaneously generate in a dose- and time-dependent manner ROS production evidenced by chemiluminescence of *Cypridina* luciferin analogue (CLA) (Figure 1A,B, Supplemenary Figure S1A). It is noteworthy that, on the contrary to CaPPs, the addition of dissolved CaCO3 (the main component of CaPPs) at 100 μg.mL−<sup>1</sup> in free MS medium did not induce ROS generation (Supplementary Figure S1B), reducing the likelihood of a chemical effect for CaPPs and providing a NP-like effect. The CaPP-induced ROS production continues to increase for about 5 h and decreases slowly after 24 h (Figure 1B). The chemiluminescence of CLA indicates the generation of superoxide anion (O2•−), and of singlet oxygen (1O2) to a lesser extent [22]. We then checked the effect of DABCO (1,4-diazabicyclo(2,2,2)octane, a scavenger of 1O2) and tiron (sodium 4,5-dihydroxybenzene-1,3-disulfonate, a scavenger of O2•−) on CaPP-induced ROS generation (Figure 1C,D). Only tiron allowed for a significant decrease of ROS generation. This suggests that CaPPs induced mainly O2•− generation in culture medium. Since hydroxyl radical (HO•) could be chemically generated from O2•− through Haber–Weiss or Fenton reactions, we further search for HO• generation by using the specific probe hydroxyphenyl fluorescein (HPF) [23]. A time-dependent increase in HPF fluorescence could be detected upon treatment with 100 μg.mL−<sup>1</sup> CaPPs (Figure 1E). This increase in HPF fluorescence was decreased by a pretreatment with 100 mM DMTU (Dimethylthiourea), a scavenger of HO• (Figure 1F) supporting the hypothesis of HO• generation.

**Figure 1.** Calcite processed particles (CaPPs)-induced ROS generation in free Murashige and Skoog (MS) medium. (**A**). Typical time and dose *Cypridina* Luminescent Analog (CLA) luminescence recorded in MS medium free of cells after addition of CaPPs. (**B**). Mean values of CaPP-induced CLA luminescence. (**C**,**D**). Effect of singlet oxygen scavenger DABCO (5 mM), and superoxide anion scavenger tiron (20 mM) on CaPP-induced CLA luminescence. The histogram represents the mean values after 20 min. (**E**). Time-dependent hydroxyphenyl fluorescein (HPF) fluorescence in response to 100 μg.mL−<sup>1</sup> CaPPs. (**F**). Effect of hydroxyl radical scavenger DMTU (100 mM) on CaPP-induced HPF fluorescence after 30 min. Data corresponded to mean values ± standard error (SE) of at least 4 independent experiments. \* Significantly different from the water treatment. Data were analyzed by variance analysis (ANOVA) and when ANOVA gave a statistically significant result, the Newman–Keuls multiple range test was used to identify which specific pairs of means were different. All numeric differences in the data were considered significantly different for a *p*-value ≤ 0.05.

#### *2.2. CaPP Particles Induced Cytosolic Calcium Variation in Tobacco BY-2 Cells*

We showed that TiO2 NPs induced a ROS-dependent increase in cytosolic calcium ([Ca<sup>2</sup>+]cyt) in BY-2 tobacco cells [15]. ROS were also shown to activate plasma membrane Ca2+ channels in plant cells [24]. We thus investigated the effect of CaPPs on cytosolic calcium level in BY-2 tobacco cultured cells expressing the Ca2<sup>+</sup>-sensitive luminescent protein aequorin in their cytosol [25]. CaPPs induced a rapid dose-dependent and transient increase in [Ca<sup>2</sup>+]cyt (Figure 2A,B). Influx of Ca2+ from the apoplast through plasma membrane was confirmed by using 500 μM La3+, a blocker of Ca2+ channels, and 3 mM EGTA, a calcium chelator (Figure 2C,D). This Ca2+ influx was dependent on the early CaPP-induced ROS production since tiron, and DMTU could also reduce the [Ca<sup>2</sup>+]cyt increase (Figure 2C,D).

**Figure 2.** CaPP-induced variations of cytosolic Ca2+ in BY-2 cells. (**A**). A typical [Ca<sup>2</sup>+]cyt variations of aequorin expressing *BY-2* cells in response to various concentrations of CaPPs. (**B**). Mean values of maximal [Ca<sup>2</sup>+]cyt increase in response to various concentrations of CaPPs. \* Significantly different from the treatment at 10 μg.mL−<sup>1</sup> CaPPs. (**C**). Effect of calcium (La<sup>3</sup>+, EGTA) and ROS (tiron and DMTU) pharmacology on 100 mg.ml−<sup>1</sup> CaPPs induced [Ca<sup>2</sup>+]cyt variations. (**D**). Mean values of maximal [Ca<sup>2</sup>+]cyt increase in response to 100 μg.mL−<sup>1</sup> of CaPPs in the presence of calcium and ROS pharmacology. Controls with pharmacology alone did not affect significantly the basal [Ca<sup>2</sup>+]cyt (not shown). Data corresponded to mean values ± SD of at least six independent experiments. \* Significantly different from the treatment at 100 μg.mL−1. Data were analyzed by variance analysis (ANOVA) and when ANOVA gave a statistically significant result, the Newman–Keuls multiple range test was used to identify which specific pairs of means were different. All numeric differences in the data were considered significantly different for a *p*-value ≤ 0.05.

Variations in [Ca<sup>2</sup>+]cyt and ROS generation are known to regulate different early events involved in signal transduction pathways such as ion channel activities and NADPH-oxidase activities induced in response to various biotic and abiotic stressors [21,26]. We then further checked if such events could be regulated by CaPPs.

#### *2.3. CaPPs Induced a NADPH Oxidase-Dependent ROS Production*

As expected from the spontaneously CaPP-induced ROS production in MS medium (Figure 1A), the chemiluminescence of CLA also rapidly increased after addition of 100 μg.mL−<sup>1</sup> CaPPs in BY-2 cell cultures (Figure 3A). From analysis of luminol-chemilumiscence, we further showed that CaPP-induced ROS generation reached a maximum at about 8 h in BY-2 cultured cells when untreated cells presented no significant increase in chemilumiscence level during the time of experiments (Figure 3B). This effect was dose-dependent (Figure 3C). The addition of 50 μM diphenyleneiodonium (DPI), an inhibitor of NADPH-oxidase [27,28], into BY-2 cell medium diminished the chemilumiscence (Figure 3C). These data sugges<sup>t</sup> the involvement of plant enzymes such NADPH-oxidase in this ROS production induced by CaPPs.

**Figure 3.** Biological CaPP-induced ROS generation by BY-2 cells. (**A**). Typical time CLA luminescence recorded with BY-2 cells after addition of 100 μg.mL−<sup>1</sup> CaPPs with or without 20 mM tiron. (**B**). Kinetic of biological ROS generation detected with luminol during 14 h after addition of 100 μg.mL−<sup>1</sup> CaPPs. (**C**). Mean values of maximal ROS increase (at 8h) in response to various CaPPs concentrations (in mg.mL−1) and in the presence 50 μM diphenyleneiodonium (DPI), an inhibitor of NADPH-oxidase. Data corresponded to mean values ± SD of at least six independent experiments. \* significantly different from the control. \*\* Significantly different from the treatment at 200 μg.mL−<sup>1</sup> CaPPs. Data were analyzed by variance analysis (ANOVA) and when ANOVA gave a statistically significant result, the Newman–Keuls multiple range test was used to identify which specific pairs of means were different. All numeric differences in the data were considered significantly different for a *p*-value ≤ 0.05.

#### *2.4. CaPPs Induce a Depolarization of Plasma Membrane Due to Anion Channel Activation*

We used an electrophysiological approach to test the effect of CaPPs on membrane potentials and ion currents of cultured cells. Upon direct addition of CaPPs, we recorded a rapid dose-dependent depolarization of BY-2 cells (Figure 4A). The depolarization was correlated with a large increase in ion currents (Figure 4B). Because impalement of a single cells could not be maintained for a long time, we further analysed the mean plasma membrane potentials and ion currents of BY-2 cell populations exposed to CaPPs for different amounts of time (Figure 4C,D). The value of the resting membrane potential (Vm) of control cells (without treatment) was around -25 mV (Figure 4C), in the same range of previous studies [26,29]. As expected from the direct addition of CaPPs (Figure 4A), cells pretreated 15 min with CaPPs were drastically depolarized (Figure 4C), but these depolarizations were transient and the cell polarizations were partly recovered for cells pretreated during 45 min (Figure 4C). These membrane potential variations were correlated with a transient increase in ion currents (Figure 4B,D) presenting the main hallmarks of anion current as previously characterized [26,29–31]. This type of current was shown to be sensitive to structurally unrelated anion channel inhibitors [26,29]. Accordingly, the increases in ion currents and the depolarizations after 15 min CaPPs pretreatment were effectively partly avoided upon pretreatment with 200 μM of glibenclamide (gli) or 9-anthracen carboxylic acid (9AC), two structurally unrelated anion channel blockers (Figure 4D), confirming

the anionic nature of these currents. These currents present the features of slow anion channels [32], but a part of the instantaneous current could be carried out by fast-activating anion channels [33]. However, these data show that increase in anion currents could be part of the early CaPP-induced signaling events.

**Figure 4.** CaPP-induced depolarization and anion current increase in BY-2 cells. (**A**). Typical depolarizations of BY-2 cell observed in response to CaPPs at 50 or 100 μg.mL−<sup>1</sup> and mean values of depolarizations. (**B**). Whole currents measured under control conditions and 5 min after addition of 100 μg.mL−<sup>1</sup> CaPPs. The protocol was as illustrated, holding potential (Vh) was Vm. Corresponding current-voltage relationships at 1.8 s. (**C**). Mean values of polarizations for BY-2 cells treated during different times with 100 μg.mL−<sup>1</sup> CaPPs and mean values of polarizations for BY-2 cells treated 15 min with 100 μg.mL−<sup>1</sup> CaPPs in the presence of 200 μM glibenclamide (gli) or 200 μM 9-antharcen carboxylic acid (9AC), two unrelated anion channel inhibitors. (**D**). Mean values of anion currents for BY-2 cells treated during different times with 100 μg.mL−<sup>1</sup> CaPPs and mean values of anion currents for BY-2 cells treated 15 min with 100 μg.mL−<sup>1</sup> CaPPs in the presence of 200 μM gli or 200 μM 9AC. Currents were recorded at −200 mV and 1.8 s of voltage clamp. Control values corresponded to the value before CaPPs addition. Data corresponded to mean values ± SD of at least six independent experiments. \* Significantly different from the control. \*\* Significantly different from the treatment at 15 min. Data were analyzed by variance analysis (ANOVA) and when ANOVA gave a statistically significant result, the Newman–Keuls multiple range test was used to identify which specific pairs of means were different. All numeric differences in the data were considered significantly different for a *p*-value ≤ 0.05.

### *2.5. CaPPs Toxicity?*

Nanoparticles were shown to induce cell death in various models [6,13,34]. We thus checked if CaPPs could induce death of BY-2 cells. No increase in cell death was observed in BY-2 cultured cells, even after 24 h of treatment (Figure 5A). We further checked if these CaPPs could have an impact on BY-2 cell culture growth. As expected from the data of cell death, addition of CaPPs in the culture medium of BY-2 cells for 7 days has no impact on the culture cell growth (Figure 5B).

**Figure 5.** CaPPs cytotoxicity in *BY-2* cultured cells. ( **A**). Cell death extent in BY-2 cultured cells detected by the Evans Blue staining after 6 or 24h of treatment with various concentrations of CaPPs. (**B**). BY-2 cultured cell growth during 7 days in the presence or not of 200 μg.mL−<sup>1</sup> CaPPs. The data corresponded to means of at least 4 independent replicates and error bars corresponded to SE.
