https://orcid.org/0000-0002-1478-7700
Figures
Abstract
Plasma technology, which can instantaneously transform air molecules into reactive species stimulating plants, potentially contributes to developing a sustainable agricultural system with high productivity and low environmental impact. In fact, plant immunity activation by exposure to a reactive gas mainly consisting of dinitrogen pentoxide (N2O5) was recently discovered, while physiological responses to N2O5 are rarely known. Here, we demonstrate early (within 10 min) physiological responses to N2O5 gas in Arabidopsis. Exposure to N2O5 gas induced an increase in cytosolic Ca2+ concentration within seconds in directly exposed leaves, followed by systemic long-distance Ca2+-based signaling within tens of seconds. In addition, jasmonic acid (JA)-related gene expression was induced within 10 minutes, and a significant upregulation of the defense-related gene PDF1.2 was observed after 1 day of exposure to N2O5 gas. These systemic resistant responses to N2O5 were found unique among air-plasma-generated species such as ozone (O3) and nitric oxide (NO)/nitrogen dioxide (NO2). Our results provide new insights into understanding of plant physiological responses to air-derived reactive species, in addition to facilitating the development of plasma applications in agriculture.
Citation: Sasaki S, Iwamoto H, Takashima K, Toyota M, Higashitani A, Kaneko T (2025) Induction of systemic resistance through calcium signaling in Arabidopsis exposed to air plasma-generated dinitrogen pentoxide. PLoS ONE 20(2): e0318757. https://doi.org/10.1371/journal.pone.0318757
Editor: Haitao Shi, Hainan University, CHINA
Received: December 4, 2024; Accepted: January 21, 2025; Published: February 6, 2025
Copyright: © 2025 Sasaki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by Frontier Research in Duo (FRiD) in Tohoku University (Project 1904). This work was also supported by JSPS KAKENHI (Grant Nos. 21K13906, 22H04947, 23H01164, and 23H01389), and the Plasma-bio Consortium (Grant Nos. 01222001, 01223002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Atmospheric pressure plasma (APP) technology, which allows for a strong non-equilibrium chemical reaction initiated by high-energy electrons, is capable of efficient chemical conversion [1–5]. Specifically, air APP can convert nitrogen (N2), oxygen (O2), and water (H2O) molecules on-site into gaseous reactive species HxNyOz [for example, ozone (O3), nitric oxide (NO), nitrogen dioxide (NO2), and hydroxyl radical (OH)], which are useful for medical [6–15], agricultural [16–21], and environmental applications [22, 23], potentially increasing the utilization of air to a great extent. Deemed a promising next-generation solution in these fields, this technology, which is capable of operating exclusively with ambient air and distributed renewable energy, such as solar power, may stand out for its environmental sustainability and versatility. In recent years, potential applications such as sterilization/virus inactivation [24–32], plant growth promotion [33–35], plant immune activation [36], nitrogen fertilization [16, 17, 20], seed germination promotion [34, 37, 38], and food preservation [39, 40] have been extensively investigated, and it has been reported that reactive oxygen species (ROS) and reactive nitrogen species (RNS) can play an important role in these processes. On the other hand, precise control of the HxNyOz gas composition is still challenging, and many studies have used mixtures of several HxNyOz (e.g., a mixture of O, O3, and HNO3). Consequently, ROS and RNS stress to plants have hardly been controlled and have not been quantified or compared; therefore, the development of the air APP applications faces the challenge of unveiled mechanism of action of APP-synthesized HxNyOz on biological targets (plants, seeds, microorganisms, etc.).
Plants are constantly subjected to a wide range of biotic and abiotic stresses from the environment [41–44]. To adapt to these stresses, plants exhibit a multitude of responses to such stresses, from gene expression to physiology and from changes in plant architecture to primary and secondary metabolism. Well-known plant responses include responses to mechanical stresses such as wounding, which change gene expression patterns and are mediated by hormones such as jasmonic acid (JA), ethylene (ET), salicylic acid (SA), and abscisic acid (ABA) [45]. More recently, our co-author discovered that systemic propagation of Ca2+ signaling from a wounded leaf is triggered within minutes by glutamate-based systems in Arabidopsis [46]. This leads to the activation of JA-induced defense responses. On the other hand, plants also respond to chemical stresses including gaseous exogenous reactive species. The plant response to gaseous O3 [47–50], for which the selective synthesis method using APP was first reported in 1857 [3], has been intensively investigated in gaseous HxNyOz. Many reports generated O3 based on APP technology and showed that O3 exposure at relatively low-density ranges (< 1 ppm) and in the long term (from hours to days) elicited various responses, including a rapid increase in cytosolic Ca2+ concentration ([Ca2+]cyt) as the earliest response and activation of salicylic acid-dependent signaling pathways [47, 51]. However, to the best of our knowledge, there are few reports on plant responses to other HxNyOz combinations, except for some reports on exogenous NO and NO2 [52–56].
We engaged in composition control and quantification of HxNyOz generated in air APP-activated gas [27, 28, 57] and reported a new APP system for the selective synthesis of dinitrogen pentoxide (N2O5) a few years ago [58], in addition to existing plasma synthesis of O3 and NO/NO2 from air. Furthermore, the activation effects of APP-synthesized N2O5 gas on plant immunity were demonstrated through pathogen inoculation tests on N2O5-stimulated plants, indicating its potential as a new technology for controlling plant diseases [59]. However, the biological mechanisms by which plants respond to dinitrogen pentoxide at the fundamental physiological level remain poorly understood. In the present study, we investigated real-time responses in plants exposed to selectively synthesized N2O5 compared to O3 and NO/NO2, focusing on Ca2+ signaling and the expression of defense-related genes. This study is the first report on the N2O5-induced local and systemic Ca2+ responses in plants, providing valuable insights into plant physiology in response to exogenous gaseous ROS and RNS, potentially useful for the development of sustainable agriculture using air APP.
Results
Construction of a plant N2O5 exposure system with live imaging of [Ca2+]cyt
N2O5 gas, which cannot be stored in ambient air, was synthesized using a newly built transportable device consisting of the plasma module (Fig 1A), two mass flow controllers, electric control components, and a mixing reactor, using the same mechanism as in the previous study [58]. The electric switches on the device determine reaction processes in the flow reactor in a mode to provide a desired gas composition consisting of N2O5, NOx (defined as a mixture of NO and NO2), and O3. The reactive gas was sprayed onto the whole-body or a single leaf of Arabidopsis thaliana plant. The local treatment of a leaf was conducted by covering the plant with a transparent film except for the targeted leaf.
(A) Experimental schematic illustration of [Ca2+]cyt imaging in plants after whole-body and local exposure to air plasma-generated reactive species. (B) Typical FT-IR absorbance spectrum of a reactive gas in the N2O5 mode. The absorbance spectra of individual species composing the best-fit synthetic spectrum for the observed spectrum are shown as negative absorbance spectra. Reactive species’ composition of reactive gases in (C) N2O5 mode, (D) NOx mode, and (E) O3 mode.
Fig 1B–1E show the gas compositions continuously generated from air are controlled at the device exit. In a reaction process mode for N2O5 generation (Fig 1C), the densities of N2O5, NO2, and O3 were approximately 230, 29, and 31 ppm, corresponding to 0.32, 0.041, and 0.044 μmol/s, respectively. In the NOx mode (Fig 1D), the densities of NO and NO2 were approximately 350 and 99 ppm, respectively, corresponding to 0.49 and 0.052 μmol/s, respectively. In O3 mode (Fig 1E), the O3 density was approximately 770 ppm, corresponding to 1.1 μmol/s. Details are provided in S1 Table. Note that the APP-synthesized N2O5 gas contained low levels of NO2 and O3 as impurities due to the unavoidable generation and decomposition reactions involved in N2O5 chemistry, and the effects of N2O5 gas should be carefully interpreted. The reactive gases mixtures in the N2O5, NOx, and O3 modes were hereafter labeled as “N2O5 gas,” “NOx gas,” and “O3 gas,” respectively. Live imaging of [Ca2+]cyt was conducted with transgenic A. thaliana expressing the GFP-based Ca2+ indicator, GCaMP3, and a fluorescence stereo microscope [44, 46, 60].
Plant damage due to exposure to reactive gases in N2O5, NOx, and O3 modes
Short-duration (10 s) exposure to O3 gas caused immediate and obvious acute injury to the plant within the first few seconds: leaves were hanging pendulously and edges were curling, giving leaves a wilted appearance, well known acute toxicity symptoms of O3 [47, 61]. At 24 hours (Fig 2A) and 2 weeks (S1 Fig) after the O3 exposure treatment, plant damage was clearly observed, and the fresh weight was significantly lower after 2 weeks (Fig 2B). In contrast, N2O5 and NOx gas exposure, despite the similar total dose (~ 10 μmol) to O3 gas exposure, showed no apparent damage after 24 hours (Fig 2A), and the fresh weight after 2 weeks was almost the same as that of the control sample exposed to dry air (Fig 2B). This clearly shows that the O3-induced toxicity to plants is very high compared to that of N2O5 and NOx. Thus, N2O5 was found less phytotoxic reactive species than O3, at least under such high-density and short-duration exposure conditions.
(A) Typical images of plants (two-week-old Col-0) at 24 hours and (B) fresh weights at 2 weeks after exposure to dry air, N2O5, NOx, and O3 gases. Statistical analysis was performed with Tukey-Kramer test (n = 7, p < 0.01). The treatment time was 30 s for dry air and N2O5, 15 s for NOx, 10 s for O3, corresponding to a total dose of approximately 10 μmol for N2O5, NO+NO2, and O3.
Ca2+ signaling induced by the APP-synthesized reactive gases in N2O5, NOx, and O3 modes
N2O5 gas exposure of the whole plant body clearly induced a gradual increase in [Ca2+]cyt after a 10-s lag period from the start of exposure, which tended to be initiated in young leaves (Fig 3, S2 Movie), whereas no significant increase in [Ca2+]cyt was observed in the dry-air treatment (Fig 3, S1 Movie). The average [Ca2+]cyt in the leaves peaked at 80 s, followed by prolonged periods (at least 5 min) of lower [Ca2+]cyt levels, although relatively high [Ca2+]cyt levels were sustained in leaf veins. The Ca2+ signal dynamics after N2O5 gas exposure was found similarly after the NOx gas exposure (S3 Movie), but the prolonged [Ca2+]cyt levels in the leaf veins tended to be higher than NOx gas. On the other hand, the O3 gas exposure resulted in a large and sharp spike in [Ca2+]cyt during the exposure and a rapid fall concurrently with leaf curling that is the acute injury symptom (S4 Movie). Based on this result (Fig 3) in light of the absence of significant toxicity associated with the present N2O5 gas exposure (Fig 2), it was concluded that the N2O5 gas exposure induced physiologically relevant Ca2+ signaling.
(A) Time-lapse images showing changes in [Ca2+]cyt and (B) time-course of changes in average [Ca2+]cyt level in the three-week-old p35S-GCaMP3 (Col-0) Arabidopsis stimulated with dry air and the plasma-generated reactive gases (N2O5, NOx, and O3). Scale bars, 10 mm.
To further investigate the systemic propagation of Ca2+ signals induced by N2O5, a targeted leaf was locally exposed to N2O5 gas (“directly exposed”), then, the non-directly exposed leaves (“indirectly exposed”) were simultaneously observed. The [Ca2+]cyt level in the “directly exposed” leaf rose rapidly during the exposure, and the Ca2+ signal subsequently propagated through the petiole to “indirectly exposed” leaves (Fig 4A, S5 Movie). The rise in [Ca2+]cyt in the directly exposed leaf was clearly larger and more rapid than that in the whole-body exposure treatment. This can be attributed to the increased dose rate of N2O5 due to the local exposure. On the other hand, the [Ca2+]cyt increase in indirectly exposed leaves tended to be slower and more persistent compared to the whole-body exposure, although the Ca2+ dynamics were varied widely among the leaves. In some indirectly exposed leaves, high [Ca2+]cyt levels were induced locally in the leaf veins, as observed in a prolonged response after the whole-body exposure treatment. Single-leaf analysis showed that, in particular, in the indirectly exposed leaves, the [Ca2+]cyt signal propagated from the petiole to the leaf blade after a lag period of approximately 1 min (Fig 4B–4E). The Ca2+ wave propagations can be roughly categorized into slow and fast propagation modes in terms of propagation speed: I. ~ 200 μm/s and II. ~ 20 μm/s. In the slow propagation (Fig 4B, 4C, S5 Movie), the Ca2+ signal propagated from the petiole to the leaf tip while avoiding the midrib. The fast propagation speed of ~ 200 μm/s was similar to previously-reported slow Ca2+ waves at 100–200 μm/s observed in the mechanical wound-induced signaling [46]. Thus, these observations show that plants have a receptible mechanism to sense N2O5 and transmit this information throughout the plant body. In addition, the N2O5-induced Ca2+ signal dynamics were partially similar to those induced by mechanical wounding, such as high [Ca2+]cyt level in the leaf vein and the propagation mode/velocity. This raises the hypothesis that the N2O5-induced leaf-to-leaf transmission employs a shared mechanism with the wound-triggered long-distance signal transmission leading to plant defense signaling [46].
(A, B) Time-lapse images showing changes in [Ca2+]cyt and (C) time-course of changes in local [Ca2+]cyt level in a plant locally stimulated with the plasma-synthesized N2O5 gas. Scale bars, 10 mm. ROI 1 to 6 were set in (B) and mean [Ca2+]cyt level in each ROI was plotted on (C).
Defense-related gene induction induced by the APP-synthesized N2O5 gas exposure
To explore the possibility of a systemic defense response as plant defense signaling induced by N2O5 gas, the expression of some defense marker genes, which were reported to be transiently upregulated in the wound-triggered systemic defense response [46], was investigated in the directly and indirectly exposed leaves as shown in Fig 5. These defense-related genes are known to be associated with the plant hormone jasmonic acid (JA) pathways. The analysis time point of 10 min was determined based on the time-course changes in the gene expression in the previous study [46]. In directly exposed leaves, the expression of JA-dependent defense marker gene [jasmonate-zim-domain protein 5 (JAZ5)] was significantly increased at 10 min (Fig 5B), indicating that the direct exposure to N2O5 gas activates a JA signaling pathway. This is consistent with the previously reported gene ontology analysis showing the induction of many JA-responsive genes with ethylene (ET) signal activation by whole body exposure to N2O5 gas [59]. In our previous study [59], transcriptome analysis by RNA-Seq revealed that the transcripts of 828 genes increased in abundance by more than two-fold in N2O5-exposed plants, and many of them were related to JA- and ET-dependent signaling pathways. Moreover, gene analysis and inoculation tests with pathogens in Arabidopsis mutants deficient in phytohormone (coi1-1, ein2-1, and npr1-1) showed the importance of JA- and ET-signaling pathways in the activation of plant immunity.
Expression of defense-related genes, including (A, D) OPR3, (B, E) JAZ5, and (C, F) JAZ7, at 10 min after (A-C) direct and (D-F) indirect exposure to the plasma-synthesized N2O5 gas. (A-C) Directly-exposed leaves and (D-F) indirectly-exposed leaves were collected at 10 min and were analyzed using qRT-PCR. Statistical analysis was performed with T-test (n = 3, *p < 0.05, **p < 0.01).
Interestingly, even in indirectly exposed leaves, significant expression of oxophytodienoate-reductase 3 (OPR3), JAZ5, and jasmonate-zim-domain protein 7 (JAZ7) was detected (Fig 5D–5F), and this expression pattern was similar to that observed in distal leaves transmitted from a directly wounded leaf [46]. This supports the hypothesis that the N2O5 induced response employs a shared mechanism with wounding-triggered long-distance transmission, which could contribute even in the N2O5-induced signal propagation to the whole body. In addition, the expression induction of JA-responsive genes shown in Fig 5 was significantly lower in direct N2O5 exposure than in indirect exposure. This suppression may be due to higher Ca2+ level and some damage overall in the directly exposed leaves (Figs 3 and 4).
The APP-synthesized N2O5 gas exposure also induced a very significant JA-responsive defense-related gene expression of plant defensin 1.2 (PDF1.2) gene, whereas other reactive gases such as O3 and NOx failed to induce (Fig 6). This raises the possibility that the observed plant disease defense response is N2O5-specific, but cannot exclude the contribution of impurities in N2O5 gas. Considering the very high sensitivity to O3 gas in Figs 2 and 3, the contribution of O3 impurity in N2O5 gas to the plant disease defense response was experimentally examined with O3 gas density approximately ten folds lower than N2O5 gas. However, exposure to the O3 gas density, adjusted to that in the APP-synthesized N2O5 gas, (called “Low O3” in the following part of this article), did not show a significant increase in PDF1.2 expression. Therefore, O3 alone in N2O5 gas is not responsible for inducing the observed PDF1.2 expression and it is suggested that N2O5 is a key species in the induced PDF1.2 expression and possibly in plant immune activation previously demonstrated through pathogen inoculation tests [59].
PDF1.2 expression in plant at 24 hours after exposure of whole body to dry air, N2O5, NOx, O3, low-density O3 (Low O3) gases. Statistical analysis was performed with Tukey-Kramer test (n = 3, p < 0.01). Treatment time was 30 s for dry air and N2O5, 15 s for dry air and NOx, 10 s for O3, corresponding to a total dose of approximately 10 μmol for N2O5, NO+NO2, and O3. Density of Low O3 at 7.9×1014 cm-3 was roughly adjusted to that (7.1×1014 cm-3) in the plasma-generated N2O5 gas as an impurity, and the supply for 30 s corresponded to approximately 1.3 μmol for Low O3.
Discussion
Plasma technology, which can convert air molecules into functional HxNyOz with low electric power consumption, could potentially contribute to the development of a sustainable agricultural system with a low environmental impact. However, the low controllability of HxNyOz synthesis using air APP and the limited understanding of plant responses to HxNyOz are ones of the reasons why plasma technology has not been fully applied in agriculture. As a first step to overcome these difficulties, the present study focused on three types of NyOz (N2O5, O3, and NOx), which can be selectively synthesized, and aimed to clarify unreported fundamental physiological responses, potentially leading to beneficial effects such as activation of plant immunity. We previously performed gene expression analysis after exposing Arabidopsis to N2O5 gas for 20 s and reported that the expression of defense response genes such as WRKY33, PAD3, and ORA59 genes and late response PDF1.2 gene was significantly induced 2 and 24 h after exposure, respectively, but these were barely or not induced in the JA signaling coi1-1 mutant [59]. Furthermore, exposure to N2O5 gas significantly reduced the size of lesions caused by Botrytis cinerea infection in wild type, ein2-1, and npr1-1 plants compared to air controls, whereas no significant difference was observed in coi1-1 [59]. Combining these previous studies with the results of the present study, we deduce that APP-synthesized N2O5 gas induces systemic Ca2+ signaling throughout the plant body, which can lead to the activation of JA signaling and plant disease responses, even when gas treatment is concentrated in a single leaf.
The APP-synthesized N2O5 gas was less toxic to plants than O3 gas (Fig 2), but more toxic than NOx gas. Exposure treatments with NOx gas did not cause significant damage to plants, even in an overdose range (> 100 μmol) where N2O5 gas caused the observed damage [59, 62]. This N2O5 gas-induced damage might be attributed to the low density of O3 as an impurity, but the toxicity symptoms (e.g., leaf yellowing rather than less leaf curling) seemed to differ from those of O3 exposure. Thus, higher doses of N2O5 may negatively affect plant health in a different manner from O3. In plants exposed to O3, a hypersensitivity response accompanied by program cell death is activated [49, 63]. The N2O5 gas exposure at the moderate dose induced a transient [Ca2+]cyt rise, the pattern of which was similar to that in NOx gas (Fig 3), but NOx gas did not induce PDF1.2 gene (Fig 6). Furthermore, the local N2O5 gas exposure of a single leaf triggered the systemic signal transmission (Fig 4), resulting in the activation of JA signaling in the distal leaves (Fig 5). High expression levels of PDF1.2 at 24 hours were found only in the N2O5 gas-treated samples among the given reactive gas exposure including the Low O3 (Fig 6), supporting that N2O5 is a key species.
The N2O5 gas-induced systemic Ca2+ signaling and the expression pattern of the defense-related genes in indirectly exposed leaves were partially similar to those in the physical wound-induced response, suggesting a shared transmission mechanism. Unlike the wound-induced response, the initial point of N2O5 attack remains still unknown, and elucidation of this is an important future challenge. The [Ca2+]cyt increase observed in this study is a relatively early response within 10 seconds of the exposure start, and is therefore an important clue toward the identification of the initial action site. A close-up observation of an N2O5 gas-exposed leaf showed that Ca2+ signaling was initiated in scatteredly distributed small spots (S2 Fig). Considering the reactivity acquisition of N2O5 on the wet surface through intermediate species (e.g., [NO2+·NO3−]aq, NO2+ aq) generation by reaction with water, the first contact of N2O5 with moist tissues, which are exposed to open air or accessible through pores such as stomata, would be key.
Two of the most studied types of induced resistance are systemic acquired resistance (SAR) and induced systemic resistance (ISR) [64]. In the SAR pathway, salicylic acid (SA) plays as a key signaling molecule and genes encoding pathogenesis-related (PR) proteins are often regarded as marker genes. It is well known that exposure of plants to O3 at relatively low-density ranges (< ppm) and over the long term (> hours) can induce the accumulation of SA and pathogenesis-related 1 (PR1) gene expression, which are important for the SAR activation pathway [47, 49]. In the present study, the induction of PR1 expression by N2O5 gas exposure for 10 s and 30 s was not significant while PDF1.2 expression was prominently increased (S3 Fig). On the other hand, ISR reportedly requires JA and ET hormonal signaling pathways. The significant induction of JA-related gene expression in the indirectly exposed leaves (Fig 5), together with the fact of JA/ET signaling pathway activation shown in the previous study [59], suggests that N2O5 gas exposure induces an ISR-like systemic response. Some reports discuss SA/JA crosstalk, where SA- and JA-mediated signaling interact with each other antagonistically or synergistically depending on the conditions [64–66]. Therefore, a clear dichotomy between SAR and ISR might be difficult, but supposing that N2O5 primarily induces JA-related signaling and SA/JA are antagonistically interfering, no significant expression of PR1 by N2O5 gas containing O3 as an impurity could be consistently explained.
The concept of controlling and supplying air-derived HxNyOz including N2O5 for plant disease management and post-harvest processes is very promising as one of plasma agricultural applications, but the progress requires further elucidation of action mechanisms of individual or mixed HxNyOz on plants. A better understanding at the molecular level of the sensing and response mechanisms of plants to unknown HxNyOz would lead to the emergence of new applications. We anticipate that results in the present study will inspire many researchers in not only the plasma applied physics and chemistry but also plant physiology and other biological fields, leading to the progress in the field of plasma agriculture.
Methods
Air plasma system for on-site generation of reactive species (N2O5, NO/NO2, O3)
The reactive gases were synthesized from air using a device developed in a previous study [58]. Briefly, the selective generation of gaseous N2O5 in extremely dry air (typically below 10 ppm of H2O) was achieved by mixing reactive gases from two independent plasma reactors: 1. low gas temperature (LT) reactor for selective O3 generation and 2. high gas temperature (HT) reactor for selective NO/NO2 generation. The setups for selective generation of N2O5, NO/NO2, and O3 were called “N2O5 mode,” “NOx mode,” and “O3 mode,” respectively. The mode can be easily changed by electric switching for the HT and LT plasma reactors. The total gas flow rate was set at 2 L/min. Further details of the device/method can be found in our previous study [58].
The composition of reactive species was analyzed using a Fourier Transform Infrared (FTIR) spectrometer (FT/IR-6100TUK; JASCO, Japan) equipped with a gas cell with five-meter-long optical path at 45°C. Reactive species’ densities were quantified using the least square error fitting of the measured spectra with synthetic spectra, composed from absorption cross sections obtained from the HITRAN database [67].
Plant cultivation and exposure of plants to reactive gases
The seeds of A. thaliana (Col-0 accession) were obtained from the Nottingham Arabidopsis Resource Centre (https://arabidopsis.info/). The transgenic line of A. thaliana expressing GCaMP3 has been described previously [46]. In brief, the GCaMP3 fragment, gifted by Loren Looger (Addgene plasmid #22692), was cloned into a pAN19 vector containing the CaMV35S promoter (p35S). The entire cassette of p35S::GCaMP3 NOSt was inserted into the plant binary vector, pBIN20. This construct was transformed into A. thaliana. A voucher specimen of this material has not yet been deposited in a publicly available herbarium. A. thaliana plants were cultured in chambers at 23°C with an 16 h-light/8 h-dark photoperiod under fluorescent lamps (100 μmol m−2 s−1). All plant experiments described in this study were performed with relevant institutional, national and international guidelines and legislation.
Reactive gases were generated by an air plasma system in each mode and supplied to 2- to 3-week-old Arabidopsis plants through a polytetrafluoroethylene (PTFE) tube with an internal diameter of 4 mm. Two-week-old plants were used for experiments to measure fresh weight after 2 weeks of gas exposure, and 3-week-old plants were used for analyses of cytoplasmic Ca2+ signals and transient gene expression. For whole-body exposure, each A. thaliana plant pot was placed 10 mm downstream from the tube exit. For local exposure, a single leaf was selectively exposed to the N2O5 gas by covering A. thaliana plant with a transparent film with a small hole.
Real-time [Ca2+]cyt imaging system
A. thaliana plants expressing genetically-encoded Ca2+ indicators were imaged with a fluorescence stereo microscope (SMZ18; Nikon, Japan) equipped with a CMOS camera (DP74; Olympus, Japan). The GFP-based Ca2+ indicator, GCaMP3, was excited using a mercury lamp (Intensilight C-HGFI; Nikon, Japan) with a 470/40 nm excitation filter and the green fluorescent signal passing through a 535/50 nm filter was acquired every 0.52 s. For local exposure, a transparent film with a small hole was used and [Ca2+]cyt observation of whole body with targeted treatment of a single leaf was realized.
The GCaMP3 signals were analysed over time at several regions of interest (ROIs) using ImageJ. Changes in [Ca2+]cyt were expressed as ΔF/F = (F − F0)/F0, where F0 represents the average baseline fluorescence determined by the average of F over the 6 frames before the exposure treatment [60].
Gene expression analysis by qRT-PCR
The analysis time points of 10 min for OPR3, JAZ5, and JAZ7 genes [46] and 24 hours for PDF1.2 and PR1 gene [59] were determined based on the time-course changes in gene expression in the previous studies. Total RNA was extracted from leaves for whole-body exposure or directly-exposed/indirectly-exposed leaves for local exposure with Trizol Reagent (Invitrogen). Finally, 500 ng of total RNA (A260/280 ratio 1.8 <, measured with NonoVue (GE Healthcare) from each sample was used for cDNA synthesis with a PrimeScript II 1st strand cDNA Synthesis Kit and random primers (TaKaRa Bio). Real-time quantitative RT-PCR was performed with SYBR Premix Ex Taq II (TaKaRa Bio), in a CFX96 Real-Time System (Bio-Rad Laboratories), with the forward and reverse primers in Table 1. For each sample condition, three additional technical iterations were performed for each of the three independent biological samples, and their means and standard deviations were calculated. The real-time RT-PCR data was normalized to Tubulin 2/3 as an internal control [68].
Supporting information
S1 Table. Reactive species’ composition of reactive gases at N2O5 mode, NOx mode, O3 mode, and low O3 mode.
https://doi.org/10.1371/journal.pone.0318757.s001
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S1 Fig. Typical images of plants at 2 weeks after exposure to dry air, N2O5, and O3 gases.
Treatment time is 30 s for dry air and N2O5, 10 s for O3, corresponding to total dose of approximately 10 μmol for N2O5 and O3.
https://doi.org/10.1371/journal.pone.0318757.s002
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S2 Fig. The close-up observation and analysis of an N2O5 gas-exposed leaf.
(A) Close-up time-lapse images showing changes in [Ca2+]cyt in the Arabidopsis plant stimulated with the N2O5 gas. Scale bars, 1 mm. (B) The difference images from the fluorescence image at 09 s. (C) Box plot of size distribution of bright spot extracted from the fluorescence image at 12 s.
https://doi.org/10.1371/journal.pone.0318757.s003
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S3 Fig.
(A) PDF1.2 and (B) PR1 expression in plant at 24 hours after exposure of whole body to dry air and N2O5 gases. Treatment time is 30 s for dry air and, 10 s and 30 s for N2O5.
https://doi.org/10.1371/journal.pone.0318757.s004
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S1 Movie. Typical time-lapse movie showing changes in [Ca2+]cyt after the whole-body exposure to dry air.
https://doi.org/10.1371/journal.pone.0318757.s005
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S2 Movie. Typical time-lapse movie showing changes in [Ca2+]cyt after the whole-body exposure to N2O5 gas.
https://doi.org/10.1371/journal.pone.0318757.s006
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S3 Movie. Typical time-lapse movie showing changes in [Ca2+]cyt after the whole-body exposure to NOx gas.
https://doi.org/10.1371/journal.pone.0318757.s007
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S4 Movie. Typical time-lapse movie showing changes in [Ca2+]cyt after the whole-body exposure to O3 gas.
https://doi.org/10.1371/journal.pone.0318757.s008
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S5 Movie. Typical time-lapse movie showing changes in [Ca2+]cyt after the local exposure to N2O5 gas.
https://doi.org/10.1371/journal.pone.0318757.s009
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