Assessing the effects of plasma-activated water on calcium signalling and arbuscular mycorrhizal symbiosis in the model legume Lotus japonicus

Assessing the effects of plasma-activated water on calcium signalling and arbuscular mycorrhizal symbiosis in the model legume Lotus japonicus Non-thermal atmospheric plasma, commonly known as cold plasma, is a weakly ionized gas, with heavy particles (such as ions, molecules, and radicals) at room temperature, while electrons possess significantly higher average energy. This thermal non-equilibrium state promotes the presence of excited chemicals, which, when they come into contact with water, generate a diverse array of reactive oxygen and nitrogen species with varying lifetimes. This diverse mixture of chemical species has demonstrated the ability to stimulate biological responses in living systems. Recently, plasma-activated water (PAW) has emerged as an environmentally friendly, cost-effective alternative that may reduce the reliance on pesticides and fertilizers in agriculture. This is due to its antimicrobial and disinfection properties, as well as its reported effects on enhancing seed germination and promoting plant growth. Furthermore, PAW has shown potential in inducing plant defense responses, effectively enhancing plant resistance against future pathogen attacks through a pre-alert state known as "priming." As research progresses, efforts are being directed towards refining this innovative "green" technology to maximize its beneficial effects within the realm of sustainable agriculture (1). Despite the growing body of literature in this field over the past few years, there is currently a lack of information regarding the impact of PAW treatment on plant-microbe symbiotic interactions in the rhizosphere. In this study, we investigated the effects of irrigating the model legume Lotus japonicus with PAW generated by a plasma torch, specifically focusing on the establishment and development of arbuscular mycorrhizal (AM) symbiosis with the AM fungus Rhizophagus irregularis. Given recent evidence demonstrating that plants sense PAW through calcium- mediated signaling pathways (2), we monitored early plant responses to various doses of PAW using L. japonicus roots expressing the bioluminescent calcium reporter aequorin, targeted to either the cytosol or nucleus. The accommodation of the AM fungus within the host plants was assessed using the Trouvelot method, which measures the frequency and intensity of root colonization, as well as the percentage of arbuscules. Chemical analyses of both PAWs (concerning hydrogen peroxide, nitrates and nitrites) and plant samples (concerning their content in total P, C, N, S) are currently underway to establish a connection between the composition of PAW and AM-mediated plant nutrient uptake. The findings of this study indicate that PAW treatments have a modulatory effect on the performance of plant AM symbiosis, which depends on the time interval of water exposure to cold plasma discharge and the duration of plant irrigation (number of weekly treatments) with PAW. By establishing a solid scientific foundation for cold plasma technology, we may gain crucial insights to develop tools and treatments aimed at increasing crop plant yields in a sustainable manner. 1) Holubová L, Kyzek S, Ďurovcová.I, Fabová.J, Horváthová.E, Ševcovicová.A, Gálová E (2020) Non-thermal plasma - A new green priming agent for plants?’’ Int. J. Mol. Sci. 21: 9466 2) Cortese.E, Settimi AG, Pettenuzzo S, Cappellin.L, Galenda.A, Famengo.A, Dabalà.M, Antoni.V, Navazio.L (2021) Plasma-activated water triggers rapid and sustained cytosolic Ca2+ elevations in Arabidopsis thaliana. Plants 10: 2516