**1. Introduction**

Several minerals have been used in agriculture [1], among which sedimentary rock that emerges from calcareous seaweed. Megagreen® is a preparation from calcite processed particles (CaPPs), elaborated from sedimentary limestone rock, which is finned and activated by a tribomecanic process [2]. These processed calcite particles are supposedly small enough to enter the leaf and have a beneficial effect on plants. The application of CaPPs on grapevines submitted to water stress was shown to increase photosynthetic CO2 fixation [3]. The benefits of CaPPs once inside the plant were supposed to be due the decomposition products, CO2 and CaO, that could feed the plant. However, the cellular responses induced by the CaPPs are poorly understood. Due to the tribomecanic processing, the CaPPs present jagged shape with some invaginations below the micrometer size (Megagreen® data sheet: https://dokumen.tips/documents/megagreen-study.html, accessed on 06/04/2020). Nanoparticles (NPs) possess a large specific surface area allowing a greater reactivity compared to macrosized particles.

Since the high surface reactivity of NPs is important for their biological effects, we hypothesised that CaPPs could have NP-like effects on plants.

Recent reviews focused on beneficial applications of nanomaterials in agricultural production [4–8]. NPs notably could induce enhancement in growth and seed yield [9], and participate in crop protection [4,10]. Although some cerium oxide nanoparticles were shown to augmen<sup>t</sup> reactive oxygen species (ROS) scavenging in *Arabidopsis thaliana* plants [11], a part of the biological effects of various NPs is proposed to be due to their ability to produce ROS, possibly due to molecular size, shape, oxidation status, increased specific surface area, bonded surface species, surface coating, solubility, and degree of aggregation and agglomeration [12–14]. We effectively showed by using *Nicotiana tabacum* L. cv. Bright Yellow 2 (BY-2) cultured cells that TiO2 NPs spontaneously generate ROS in the culture medium, but also induced a rapid biological ROS production and a ROS-dependent increase in cytosolic calcium ([Ca<sup>2</sup>+]cyt) [15]. Variations of [Ca<sup>2</sup>+]cyt serve as secondary messenger involved in many adaptation and developmental processes in plants [16,17]. Reactive oxygen species also play a key signal transduction role in plant cells, such as growth regulation, development, responses to environmental stimuli and cell death [18,19]. However, the response of plants to NPs varies with the growth stages, type of plant species and the nature of NPs. Thus, they could have positive and negative effects on plants [20]. In this study, we tested the impact of CaPPs on cell viability and further checked if CaPPs as NPs could induce ROS generation due to their increased specific surface area and carried out an experimental layout on plant cultured cells to study the impact of CaPPs on variations of [Ca<sup>2</sup>+]cyt, biological ROS generation and ion fluxes variations, early cellular responses frequently involved in signalling processes [21].
