Calcium-Based Systemic Activation of Plant Defense Response by Exposure to N₂O₅ Gas Synthesized in Atmospheric-Pressure Plasma Technology

Atmospheric-pressure plasma (APP) technology, enabling to convert air molecules into multi-functional reactive species [e.g., reactive oxygen and nitrogen species (RONS)] with electricity, is of great interest and has been extensively investigated. For example, ozone (0₃) produced by the APP technology has multifunctional abilities such as disinfection and deodorizing, and has already been into practical use [1]. Recently, we have developed a new air APP device/method that allows highly selective production of dinitrogen pentoxide (N₂O₅) exclusively from only air and electricity sources [2]. N₂O₅, a unique oxidizing and nitrating compound, is a promising chemical for a variety of applications, but requires multiple dangerous raw materials (careful handling) to synthesize N₂O₅ by conventional methods. Therefore, it has not yet been used for bio-applications. Our APP device/method for N₂O₅ production does not require careful handling, complicated manufacturing equipment, and toxic substances, and have the potential to be used in scientific and industrial applications. Here, we investigated the applicability of APP-generated N₂O₅ in agriculture.<br/>0₃ exposure has been reported to result in expression of a number of defense-related genes that are also induced during a hypersensitive response (HR) in plants. In addition, 0₃ exposure allows systemic acquire resistance (SAR) in the phytohormone salicylic acid-mediated defense response, which generally causes growth retardation and visible damage [3]. On the other hands, we found that exposure to APP-generated N₂O₅ induces plant defensin genes such as PDF1.2 that follow a salicylic acid-independent pathway as well as no-growth retardation. We also found that using <i>Arabidopsis thaliana</i>, which expresses a GCaMP3 fluorescent protein-based cytosolic Ca²⁺ sensor, the cytosolic calcium ion concentration ([Ca²⁺]_cyt) increased within 10 sec of exposure to ~200ppm of APP-generated N₂O₅. Furthermore, local N₂O₅ exposure to a single leaf using a clear plastic film induced an increase in [Ca²⁺]_cyt that spreads not only to directly exposed leaf but also to indirectly exposed leaves. This response is very similar to wound-induced [Ca²⁺]_cyt signaling, which elicits a systemic defense response by the plant hormone jasmonic acid (JA) [4]. To verify the similarity of the reactions, the expression of JA-related genes was monitored in leaves exposed to the APP-generated N₂O_5.RT-PCR results showed that expression of JA-related genes was significantly induced in both directly and indirectly exposed leaves. These results indicate that the APP-generated N₂O₅ exposure, unlike ozone, provokes an induced systemic resistance without growth retardation. The presentation will also discuss the applicability of APP technology in agriculture.<br/>[1] M. Remondino and L. Valdenassi, Different Uses of Ozone: Environmental and Corporate Sustainability. Literature Review and Case Study, Sustainability <b>10</b>, 4783 (2018).<br/>[2] S. Sasaki, K. Takashima, and T. Kaneko, Portable Plasma Device for Electric N₂O₅ Production from Air, Ind. Eng. Chem. Res., <b>60</b>, 798 (2021)<br/>[3] Y.K. Sharma, et al., Ozone-induced responses in <i>Arabidopsis thaliana</i>: The role of salicylic acid in the accumulation of defense-related transcripts and induced resistance. Proc. Natl. Acad. Sci. USA <b>93, </b>5099 (1996).<br/>[4] M. Toyota, et al., Glutamate triggers long-distance, calcium-based plant defense signaling, Science, <b>361</b>, 1112 (2018).