Real-time live imaging of cytosolic hydrogen peroxide and Ca2+ of Marchantia polymorpha gemmalings reveal immediate initial responses of plant cells triggered by nonthermal plasma irradiation

Cold atmospheric pressure plasma generators capable of generating plasma under normal pressure and temperature conditions have recently been developed, and their biological applications have been extensively studied. Plasma irradiation has been reported to affect plant germination and growth; however, the molecular mechanism underlying these effects and initial cellular responses to plasma irradiation remains poorly understood. To unravel the molecular and cellular mechanisms underlying the effects of plasma irradiation on plants, we have been establishing novel experimental systems using a model liverwort Marchantia polymorpha. We here focused on the initial responses of plant cells to plasma irradiation. To investigate immediate cellular responses following plasma irradiation, we developed a new plasma device that allows irradiation under a microscope. Through integration with live fluorescence imaging, we established an experimental setup to track, the dynamics of intracellular concentration of H₂O₂ and Ca²⁺ as representative initial cellular responses. We revealed that plasma irradiation induced a rapid and transient increase in intracellular concentration of H₂O₂ and Ca²⁺ in Marchantia gemmalings. Pharmacological analyses suggested that the long-lived reactive species, H₂O₂, generated by the plasma generator was directly delivered into the plant cells. Competitive inhibitors of Ca²⁺ channels abolished the Ca²⁺ rise, suggesting that plasma irradiation immediately activate plasma membrane Ca²⁺ channel(s) to induce Ca²⁺ influx. Importantly, this study marks the inaugural demonstration of real-time monitoring of cytosolic H₂O₂ and Ca²⁺ dynamics in plants, triggered by plasma irradiation.