**3. Discussion**

The CaPPs application has been shown to be beneficial on several crops such as olive trees, maize, strawberry and lettuce, especially under drought conditions (technical data sheet for Megagreen ®: https://dokumen.tips/documents/megagreen-study.html, accessed on 06/04/2020). The benefits of CaPPs once inside the leaves was attributed to the decomposition products CO2 and CaO that could feed the plant. CaPPs application on grapevines subjected to water stress was shown to increase photosynthetic CO2 fixation [3]. The CaPPs penetrating directly into the leaves are supposed to increase CO2 saturation in the leave leading to stomatal closure and therefore a reduction in evapotranspiration a reduction of photorespiration in favor of photosynthesis [3]. Spray of CaO were also shown to correct Ca2+ deficiency in groundnut [35] although the mean levels of Ca2+ were not statistically di fferent between CaPP-treated and untreated vines [36]. However, due to the size distribution of these CaPPs ranging from the nano- to the microparticle (0.1 to 20 μm), we hypothesized NP-like e ffects of CaPPs at the cellular level. By using nonphotosynthetic BY-2 cultured plant cells, we could discriminate the effects of NPs from already-reported e ffects on photosynthetic activity.

Our data showed that CaPPs induce ROS generation independently of any living cells. This ROS production is dose- and time-dependent and seemed to be mainly due to O2•− (detected by CLA and scavenged by tiron) and subsequently HO• (detected by HPF and scavenged by DMTU) through Haber–Weiss or Fenton reactions after the dismutation of O2•− into H2O2. These data correlate with previous one indicating that NPs from di fferent nature can produce ROS due to their increased specific surface area [12–14].

Our pharmacological data with ROS scavengers show that these CaPP-induced ROS could be responsible in BY-2 cells for the induction of well-known cellular events involved in the signalling process, such as calcium influx through plasma membrane Ca2+ channels, subsequent NADPH-oxidase stimulation and anion channel activation. The NADPH-oxidase stimulation and anion channel activations could also be recorded in response to CaPPs in *A. thaliana* cultured cells (Supplementary Figure S2). ROS generation and the cytosolic calcium increase are reminiscent with what was observed in response to TiO2 NPs in BY-2 cells [15], or in response to ZnO NPs in *Salicornia* [37], but also in responses to O3, another oxidative stress [38], on tobacco cells [39] and *A. thaliana* cultured cells [21,40–42]. Less data are available on the e ffect of NPs on ion channel regulation especially in plants, but it is noticeable that polystyrene NPs could activate CFTR-Cl− channels in hamster kidney cells [43] and O3 anion currents in *A. thaliana* cells [21].

Although CaPPs do not seem to be toxic for BY-2 cells, such signalling events are frequently related to the induction of programmed cell death (PCD) [21,26,29]. E ffectively we could observe in *A. thaliana* cells after addition of CaPPs an increase in cell death slowing the whole suspension growth (Supplementary Figure S3). Toxic e ffects of nanoparticles were already observed in response to various NPs such as ZnONPs or AgNPs in algae [44,45] or CuONPs, SiNPs and single-wall carbon nanotubes

on a terrestrial model [34,46,47], sometimes due to the PCD process [34]. In *A. thaliana,* cell death was dependent on transcription and translation (Figure S3), e ffectively suggesting an active process, thus a PCD. The discrepancy observed in terms of cell death between the two cultured cell lines, since there was no record of cell death or the slowing of suspension cell growth for BY-2 cells, which could be explained by a di fference in sensitivity. E ffectively, carbon nanotubes were shown to induce the growth enhancement of tobacco cells [48] when they induce PCD in *A. thaliana* and rice [14,34]. However, the CaPP-induced PCD in *A. thaliana* cells could be reduced by the ROS scavengers DMTU and tiron, the blockers of Ca2+ influx, BAPTA and La3<sup>+</sup> and the anion channel blockers 9AC and glibenclamide (Supplementary Figure S3). These data support the hypothesis that the CaPP-induced ROS generation induces the signaling pathways leading to the PCD process. It is also noteworthy that these cellular events are also involved in stomatal aperture regulation [49]. We could further confirm the decrease of stomatal aperture 30 min after application of 50 μM CaPPs on the epidermis *A. thaliana* leaves (Supplementary Figure S4). Thus, the CaPP-induced stomatal closure could be due to not only an increase in CO2 saturation of the leaves [3], but also to the CaPP-induced ROS generation.

In summary, our study shows that CaPPs could have, in addition to its known e ffects on photosynthesis [3], NP-like e ffects due to their size distribution. The abiotic ROS generation induced by these CaPPs could induce cellular events that could be involved in various signaling pathways. More studies, particularly with di fferent species, will be needed to clarify the possible outputs of these signaling pathways.

#### **4. Materials and Methods**

### *4.1. CaPP Particles*

Megagreen ® is composed of calcite processed particles (CaPPs) elaborated from sedimentary limestones rock, which is finned and activated by tribomecanic process (European Patent N◦ WO/2000/064586). These CaPPs present a distribution ranging from the nano- to the microparticle (0.1 to 20 μm). The chemical composition of CaPPs is: total calcium carbonate 823.0 <sup>g</sup>·kg−1; SiO2 85.2 g kg−1; MgO 30.2 <sup>g</sup>·kg−1; Fe 8.78 <sup>g</sup>·kg−1, and other trace elements. CaPPs were diluted in distilled water and pH adjusted to 5.8 with HCl.

#### *4.2. Plant Cell Culture Conditions*

*Nicotiana tabacum* BY-2 cultured cells were grown in Murashige and Skoog medium (MS medium) [50] complemented with 30 g.L−<sup>1</sup> sucrose, 0.1 mg.L−<sup>1</sup> 2,4 D (pH 5.8) and maintained by weekly dilution (2/80). The cell culture was agitated on a rotary shaker at 120 rpm at 22 ± 2 ◦C in the dark. Such cells are white and nonphotosynthetic. All experiments were performed at 22 ± 2 ◦C using the cells in log-phase (6 days after subculturing).

Cell growth was estimated for by recording each day after subculture the fresh weight of cells contained in 50 mL of culture for BY-2 cell cultures.

#### *4.3. Monitoring of ROS Production*

The production of ROS was monitored using different techniques and probes. The chemiluminescence of the *Cypridina* luciferin analog (CLA) react mainly with O2•− and 1O2 with light emission [22]. Chemiluminescence from CLA was monitored using a FB12-Berthold luminometer (with a signal integrating time of 0.2 s). For data analysis, the luminescence ratio (L/Lbasal) was calculated by dividing the luminescence intensities of CLA-luminescence (L) with the luminescence intensity before treatment (Lbasal). Hydroxy radicals (HO•) formation was also checked using the specific probe hydroxyphenyl fluorescein (HPF) [23]. Briefly, HPF was added to 1 mL of MS medium to a final concentration of 10 μM at di fferent times after the addition of 100 mg.mL−<sup>1</sup> of CaPPs. The fluorescence increase was monitored at 515 nm after an excitation at 490 nm using a F-2000 spectrofluorimeter (Hitachi, Tokyo, Japan).

#### *Int. J. Mol. Sci.* **2020**, *21*, 4279

For biological production of ROS, we used the chemiluminescence of luminol [51], which is dependent on the activity of cell-derived peroxidase. Briefly, 6 mL of the cultured cells were inoculated with CaPPs. Before each measurement, 200 μL of the cell culture was added prior to the addition of 5 μL luminol (1.1 mM). Chemiluminescence measurements were carried out at 30 min intervals using a FB12-Berthold luminometer (signal integrating time 0.2 s).

#### *4.4. Aequorin Luminescence Measurements*

Cytoplasmic Ca2+ variations were recorded from BY-2 cultured cells expressing the apoaequorin gene [25]. For Ca2+ measurement, aequorin was reconstituted by an overnight incubation of the cell cultures in MS medium supplemented with 2.5 μM native coelenterazine. Cell culture aliquots (450 μL in MS medium) were transferred carefully to a luminometer glass tube and luminescence was recorded continuously at 0.2 s intervals using a FB12-Berthold luminometer (Berthold Technologies, Bad Wildbad, Germany). Treatments were performed by 50 μL injections containing the CaPPs. At the end of each experiment, residual aequorin was discharged by addition of 500 μL of a 1M CaCl2 solution dissolved in 100% methanol. The resulting luminescence was used to estimate the total amount of aequorin in each experiment. Calibration of the calcium measurement was performed using the equation: pCa = 0.332588(−logk) +5.5593, where k is a rate constant equal to luminescence counts per second divided by total remaining counts [25]. To test the e ffects of each di fferent pharmacological treatment, BY-2 cells were pretreated for 15 min before the application of CaPPs.
