*4.5. Electrophysiology*

Experiments were conducted on BY-2 cells maintained in their culture medium to limit stress (main ions in MS medium 28 mM NO3 − and 16 mM K<sup>+</sup>) [26]. Individual cells were immobilized by a microfunnel (approximately 30 to 80 μm outer diameter and controlled by a micromanipulator (WR6-1, Narishige, Tokyo, Japan). Impalement were carried out with a piezoelectric micromanipulator (PCS-5000, Burleigh Inst., New York, NY, USA) in a chamber (500 μL) made of Perspex. Voltage-clamp measurements of whole-cell currents from intact BY-2 cells presenting stable running membrane potential were carried out at room temperature (20–22 ◦C) using the technique of the discontinuous single voltage-clamp microelectrode [52] adapted to plant cells [40,53]. Microelectrodes were made from borosilicate capillary glass (Clark GC 150F, Clark Electromedical, Pangbourne Reading, UK) pulled on a vertical puller (Narishige PEII, Tokyo, Japan). Their tips were less than 1 μm diameter; they were filled with 600 mM KCl, and had electrical resistances between 20 and 50 M Ω with the culture medium. Specific software (pCLAMP 8) drives the voltage clamp amplifier (Axoclamp 2A, Molecular Devices, Sunnyvale, CAL, USA). Voltage and current were digitalised with a Digidata 1322A (Molecular Devices, Sunnyvale, CAL, USA). In whole-cell current measurements the membrane potential was held to the value of the resting membrane potential. Current recordings were obtained by hyperpolarizing pulses from −200 to +80 mV (20 mV, 2 s steps of current injection, 6s of settling time). We systematically checked that cells were correctly clamped by comparing the protocol voltage values with those really imposed. Only microelectrodes presenting a linear relationship were used.

#### *4.6. Cell Viability Assays*

Cell viability was checked using the vital dye, Evans Blue. Cells (50 μL) were incubated for 5 min in 1 mL phosphate bu ffer pH 7 supplemented with Evans blue to a final concentration of 0.005%.<sup>21</sup> Cells that accumulated Evans blue were considered dead. At least 1000 cells were counted for each independent treatment. The experiment was repeated at least 4 times for each condition.

### *4.7. Statistical Analysis*

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

means were di fferent. All numeric di fferences in the data were considered significantly di fferent for a *p*-value ≤ 0.05.

**Supplementary Materials:** Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/12/4279/ s1. Figure S1. A. Mean values of CaPP-induced CLA luminescence B. Comparison of CaPP- and CaCO3-induced ROS generation in free MS medium. Figure S2: **A.** Kinetic of biological ROS generation detected with luminol during 7 h after addition of 100 μg.mL−<sup>1</sup> CaPPs. **B.** Mean values of polarizations for A. thaliana cells treated during di fferent times with 100 μg.mL−<sup>1</sup> CaPPs and mean values of polarizations for A. thaliana cells treated 15 min with 100 μg.mL−<sup>1</sup> CaPPs in presence of 200 μM glibenclamide (gli) or 200 μM 9-antharcen carboxylic acid (9AC), two unrelated anion channel inhibitors. Figure S3: A. Dose-dependent cell death reaching about 50% of the *Arabidopsis thaliana* cell population was observed after 6 h after treatment with 200 μg. mL−<sup>1</sup> CaPPs. B. Decrease of the culture growth induced by 200 μg. mL−<sup>1</sup> CaPPs. C. Decrease of cell death extent by pretreatments with actinomycin D (AD, 20 μg/mL), cycloheximide (Chx, 20 μg/mL), inhibitors of traduction and translation, ROS scavengers Tiron (5 mM) and DMTU (100 mM), Ca2+ channel blocker La3<sup>+</sup> (500 μM), Ca2+ chelator, BAPTA (3 mM), and anion channel blockers, glibenclamide (gli 200 μM) and 9AC (200 μM). For each pretreatment, cells were incubated for 15 min before CaPPs treatment. Figure S4: Applications of 100 μg mL−<sup>1</sup> CaPPs reduce the stomatal aperture of *A. thaliana* leaves. Figure S4: Applications of 100 μg.mL−<sup>1</sup> CaPPs reduce the stomatal aperture of *A. thaliana* leaves. In presence of 3 mM EGTA, the CaPPs-induced stomatal closure was reduced.

**Author Contributions:** D.T., T.Z., D.A.-B., T.K. (Takashi Kadono), R.E. and P.M. carried out the experiments. S.C. helped maintaining the cultures. F.B. supervised the project with the help of R.E. and T.K. (Tomonori Kawano), F.B. wrote the manuscript with support from D.T. The manuscript was written through contributions of all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by funds from Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation to LIED.

**Acknowledgments:** The authors thank Faouzi Attia, Joël Briand and Bernadette Biligui for their implication at early step of the project, and Agronutrition for providing us Megagreen ®. This study was supported by funds from Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation to LIED.

**Conflicts of Interest:** The authors declare no conflict of interest
