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Non-thermal plasma technology is increasingly being applied in the plant biology field. Despite the variety of beneficial effects of plasma-activated water (PAW) on plants, information about the mechanisms of PAW sensing by plants is still limited. In this study, in order to link PAW perception to the positive downstream responses of plants, transgenic Arabidopsis thaliana seedlings expressing the Ca 2+ -sensitive photoprotein aequorin in the cytosol were challenged with water activated by low-power non-thermal plasma generated by a dielectric barrier discharge (DBD) source. PAW sensing by plants resulted in the occurrence of cytosolic Ca 2+ signals, whose kinetic parameters were found to strictly depend on the operational conditions of the plasma device and thus on the corresponding mixture of chemical species contained in the PAW. In particular, we highlighted the effect on the intracellular Ca 2+ signals of low doses of DBD-PAW chemicals and also presented the effects of consecutive plant treatments. The results were discussed in terms of the possibility of using PAW-triggered Ca 2+ signatures as benchmarks to accurately modulate the chemical composition of PAW in order to induce environmental stress resilience in plants, thus paving the way for further applications in agriculture.

Increasing evidence indicates that water activated by plasma discharge, termed as plasma-activated water (PAW), can promote plant growth and enhance plant defence responses. Nevertheless, the signalling pathways activated in plants in response to PAW are still largely unknown. In this work, we analysed the potential involvement of calcium as an intracellular messenger in the transduction of PAW by plants. To this aim, Arabidopsis thaliana (Arabidopsis) seedlings stably expressing the bioluminescent Ca 2+ reporter aequorin in the cytosol were challenged with PAW generated by a plasma torch. Ca 2+ measurement assays demonstrated the induction by PAW of rapid and sustained cytosolic Ca 2+ elevations in Arabidopsis seedlings. The dynamics of the recorded Ca 2+ signals were found to depend upon different parameters, such as the operational conditions of the torch, PAW storage, and dilution. The separate administration of nitrate, nitrite, and hydrogen peroxide at the same doses as those measured in the PAW did not trigger any detectable Ca 2+ changes, suggesting that the unique mixture of different reactive chemical species contained in the PAW is responsible for the specific Ca 2+ signatures. Unveiling the signalling mechanisms underlying plant perception of PAW may allow to finely tune its generation for applications in agriculture, with potential advantages in the perspective of a more sustainable agriculture.