Utilizing plasma-generated N2O5 gas from atmospheric air as a novel gaseous nitrogen source for plants.

Fixing atmospheric nitrogen for use as fertilizer is a crucial process in promoting plant growth and enhancing crop yields in agricultural production. Currently, the chemical production of nitrogen fertilizer from atmospheric N2 relies on the energy-intensive Haber-Bosch process. Therefore, developing a low-cost and easily applicable method for fixing nitrogen from the air would provide a beneficial alternative. In this study, we tested the utilization of dinitrogen pentoxide (N2O5) gas, generated from oxygen and nitrogen present in ambient air with the help of a portable plasma device, as a nitrogen source for the model plant Arabidopsis thaliana. Nitrogen-deficient plants supplied with medium treated with N2O5, were able to overcome nitrogen deficiency, similar to those provided with medium containing a conventional nitrogen source. However, prolonged direct exposure of plants to N2O5 gas adversely affected their growth. Short-time exposure of plants to N2O5 gas mitigated its toxicity and was able to support growth. Moreover, when the exposure of N2O5 and the contact with plants were physically separated, plants cultured under nitrogen deficiency were able to grow. This study shows that N2O5 gas generated from atmospheric nitrogen can be used as an effective nutrient for plants, indicating its potential to serve as an alternative nitrogen fertilization method for promoting plant growth.

Atmospheric-pressure plasma irradiation can disrupt tobacco mosaic virus particles and RNAs to inactivate their infectivity.

Low-temperature atmospheric-pressure air plasma is a source of charged and neutral gas species. In this study, N-carrying tobacco plants were inoculated with plasma irradiated and non-irradiated tobacco mosaic virus (TMV) solution, resulting in necrotic local lesions on non-irradiated, but not on irradiated, TMV-inoculated leaves. Virus particles were disrupted by plasma irradiation in an exposure-dependent manner, but the viral coat protein subunit was not. TMV RNA was also fragmented in a time-dependent manner. These results indicate that plasma irradiation of TMV can collapse viral particles to the subunit level, degrading TMV RNA and thereby leading to a loss of infectivity.

Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization.

Gas-liquid interfacial atmospheric-pressure plasma jets (GLI-APPJ) are used medically for plasma-induced cell-membrane permeabilization. In an attempt to identify the dominant factors induced by GLI-APPJ responsible for enhancing cell-membrane permeability, the concentration and distribution of plasma-produced reactive species in the gas and liquid phase regions are measured. These reactive species are classified in terms of their life-span: long-lived (e.g., H2O2), short-lived (e.g., O2•-), and extremely-short-lived (e.g., •OH). The concentration of plasma-produced •OHaq in the liquid phase region decreases with an increase in solution thickness (<1 mm), and plasma-induced cell-membrane permeabilization is found to decay markedly as the thickness of the solution increases. Furthermore, the horizontally center-localized distribution of •OHaq, resulting from the center-peaked distribution of •OH in the gas phase region, corresponds with the distribution of the permeabilized cells upon APPJ irradiation, whereas the overall plasma-produced oxidizing species such as H2O2aq in solution exhibit a doughnut-shaped horizontal distribution. These results suggest that •OHaq is likely one of the dominant factors responsible for plasma-induced cell-membrane permeabilization.

Increase of secondary metabolites in sweet basil (Ocimum basilicum L.) leaves by exposure to N2O5 with plasma technology.

Exposure to N2O5 generated by plasma technology activates immunity in Arabidopsis through tryptophan metabolites. However, little is known about the effects of N2O5 exposure on other plant species. Sweet basil synthesizes many valuable secondary metabolites in its leaves. Therefore, metabolomic analyses were performed at three different exposure levels [9.7 (Ex1), 19.4 (Ex2) and 29.1 (Ex3) µmol] to assess the effects of N2O5 on basil leaves. As a result, cinnamaldehyde and phenolic acids increased with increasing doses. Certain flavonoids, columbianetin, and caryophyllene oxide increased with lower Ex1 exposure, cineole and methyl eugenol increased with moderate Ex2 exposure and L-glutathione GSH also increased with higher Ex3 exposure. Furthermore, gene expression analysis by quantitative RT-PCR showed that certain genes involved in the syntheses of secondary metabolites and jasmonic acid were significantly up-regulated early after N2O5 exposure. These results suggest that N2O5 exposure increases several valuable secondary metabolites in sweet basil leaves via plant defense responses in a controllable system.

Activation of plant immunity by exposure to dinitrogen pentoxide gas generated from air using plasma technology.

Reactive nitrogen species (RNS) play an important role in plant immunity as signaling factors. We previously developed a plasma technology to partially convert air molecules into dinitrogen pentoxide (N2O5), an RNS whose physiological action is poorly understood. To reveal the function of N2O5 gas in plant immunity, Arabidopsis thaliana was exposed to plasma-generated N2O5 gas once (20 s) per day for 3 days, and inoculated with Botrytis cinerea, Pseudomonas syringae pv. tomato DC3000 (Pst), or cucumber mosaic virus strain yellow (CMV(Y)) at 24 h after the final N2O5 gas exposure. Lesion size with B. cinerea infection was significantly (P < 0.05) reduced by exposure to N2O5 gas. Propagation of CMV(Y) was suppressed in plants exposed to N2O5 gas compared with plants exposed to the air control. However, proliferation of Pst in the N2O5-gas-exposed plants was almost the same as in the air control plants. These results suggested that N2O5 gas exposure could control plant disease depending on the type of pathogen. Furthermore, changes in gene expression at 24 h after the final N2O5 gas exposure were analyzed by RNA-Seq. Based on the gene ontology analysis, jasmonic acid and ethylene signaling pathways were activated by exposure of Arabidopsis plants to N2O5 gas. A time course experiment with qRT-PCR revealed that the mRNA expression of the transcription factor genes, WRKY25, WRKY26, WRKY33, and genes for tryptophan metabolic enzymes, CYP71A12, CYP71A13, PEN2, and PAD3, was transiently induced by exposure to N2O5 gas once for 20 s peaking at 1-3 h post-exposure. However, the expression of PDF1.2 was enhanced beginning from 6 h after exposure and its high expression was maintained until 24-48 h later. Thus, enhanced tryptophan metabolism leading to the synthesis of antimicrobial substances such as camalexin and antimicrobial peptides might have contributed to the N2O5-gas-induced disease resistance.

Idiopathic thrombocytopenia in Japanese black cattle.

A female Japanese Black cow showed hemorrhage under the mucous membrane of the eye conjunctiva and the vagina, and thrombocytopenia in peripheral blood. The clinical manifestations repeated spontaneously during long-term observation. The clinical findings were stable except for severe periodic bleeding. Prothrombin time, activated partial thromboplastin time and fibrinogen levels were normal. The anti-platelet antibody in the serum was analyzed by indirect platelet coagulation tests. Because the platelets in the micro-plate wells of the subject series showed coagulation up to 1:128, it was judged that anti-platelet antibody was present in the serum. A number of megakaryocytes were recognized in smear samples obtained from bone marrow of the scapula immediately after euthanasia. Therefore, this case was diagnosed as idiopathic thrombocytopenia.