# Changes in Gene Expression After Exposing Arabidopsis thaliana Plants to Nanosecond High Amplitude Electromagnetic Field Pulses

Alexis Porcher, Nancy Wilmot, Pierre Bonnet, Vincent Procaccio, Alain Vian

► To cite this version:

Alexis Porcher, Nancy Wilmot, Pierre Bonnet, Vincent Procaccio, Alain Vian. Changes in Gene Expression After Exposing Arabidopsis thaliana Plants to Nanosecond High Amplitude Electromagnetic Field Pulses. Bioelectromagnetics, 2023, 45 (1), pp.4-15. 10.1002/bem.22475. hal-04494354

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# Changes in Gene Expression After Exposing Arabidopsis thaliana Plants to Nanosecond High Amplitude Electromagnetic Field Pulses

Alexis Porcher, Nancy Wilmot, Pierre Bonnet, Vincent Procaccio, Alain Vian

► To cite this version:

Alexis Porcher, Nancy Wilmot, Pierre Bonnet, Vincent Procaccio, Alain Vian. Changes in Gene Expression After Exposing Arabidopsis thaliana Plants to Nanosecond High Amplitude Electromagnetic Field Pulses. Bioelectromagnetics, 2024, 45 (1), pp.4-15. 10.1002/bem.22475. hal-04494354

HAL Id: hal-04494354

https://hal.inrae.fr/hal-04494354v1

Submitted on 7 Mar 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Changes in Gene Expression After Exposing *Arabidopsis thaliana* Plants to Nanosecond High Amplitude Electromagnetic Field Pulses

Alexis Porcher,¹ Nancy Wilmot,² Pierre Bonnet,¹ Vincent Procaccio,² and Alain Vian D³*

¹Université Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Pascal, Clermont-Ferrand, France

²Univ Angers, CHU Angers, INSERM, CNRS, MITOVASC, SFR ICAT, Angers, France

³Univ Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, Angers, France

The biological effects of exposure to electromagnetic fields due to wireless technologies and connected devices are a subject of particular research interest. Ultrashort high-amplitude electromagnetic field pulses delivered to biological samples using immersed electrodes in a dedicated cuvette have widely demonstrated their effectiveness in triggering several cell responses including increased cytosolic calcium concentration and reactive oxygen species (ROS) production. In contrast, the effects of these pulses are poorly documented when electromagnetic pulses are delivered through an antenna. Here we exposed *Arabidopsis thaliana* plants to 30,000 pulses (237 kV m⁻¹, 280 ps rise-time, duration of 500 ps) emitted through a Koshelev antenna and monitored the consequences of electromagnetic fields exposure on the expression levels of several key genes involved in calcium metabolism, signal transduction, ROS, and energy status. We found that this treatment was mostly unable to trigger significant changes in the messenger RNA accumulation of calmodulin, Zinc-Finger protein ZAT12, NADPH oxidase/respiratory burst oxidase homolog (RBOH) isoforms D and F, Catalase (CAT2), glutamate-cystein ligase (GSH1), glutathione synthetase (GSH2), Sucrose non-fermenting-related Kinase 1 (SnRK1) and Target of rapamycin (TOR). In contrast, Ascorbate peroxidases APX-1 and APX-6 were significantly induced 3 h after the exposure. These results suggest that this treatment, although quite strong in amplitude, is mostly ineffective in inducing biological effects at the transcriptional level when delivered by an antenna. Bioelectromagnetics. 45:4–15, 2024. © 2023 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.

**Keywords:** *Arabidopsis thaliana*; Koshelev antennae; nanosecond electromagnetic pulses; gene expression; ascorbate peroxidase

## INTRODUCTION

Electromagnetic fields of various frequencies are increasingly frequent in the environment due to the fast development of wireless telecommunications and many connected objects using continuous wave (CW) or pulsed magnetic field (PMF) of high frequency within the range of 1–3 GHz. This has raised concerns and questions about their possible interactions with living cells and organisms [Cucurachi et al., 2013], including plants [Vian et al., 2016; Kaur et al., 2021]. Living tissues are indeed poor dielectrics and their interactions with electromagnetic field cause a temperature elevation

Grant sponsor: LRC BIOEM ("Laboratoire de Recherche Conventionné), grant number: LRC No. 002-2011.

Conflicts of interest: None.

*Correspondence to: Alain Vian, Univ Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, F-49000 Angers, France.
E-mail: alain.vian@univ-angers.fr

Received for review 2 September 2021; Revised 17 March 2023; Accepted 23 May 2023

DOI:10.1002/bem.22475

Published online 6 July 2023 in Wiley Online Library (wileyonlinelibrary.com).

<footnote>This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.</footnote>
# Changes in Gene Expression After Exposing *Arabidopsis thaliana* Plants to Nanosecond High Amplitude Electromagnetic Field Pulses

Alexis Porcher,<fnref n="1" /> Nancy Wilmot,<fnref n="2" /> Pierre Bonnet,<fnref n="1" /> Vincent Procaccio,<fnref n="2" /> and Alain Vian ID<fnref n="3" />*

The biological effects of exposure to electromagnetic fields due to wireless technologies and connected devices are a subject of particular research interest. Ultrashort high-amplitude electromagnetic field pulses delivered to biological samples using immersed electrodes in a dedicated cuvette have widely demonstrated their effectiveness in triggering several cell responses including increased cytosolic calcium concentration and reactive oxygen species (ROS) production. In contrast, the effects of these pulses are poorly documented when electromagnetic pulses are delivered through an antenna. Here we exposed *Arabidopsis thaliana* plants to 30,000 pulses (237 kV m⁻<fnref n="1" />, 280 ps rise-time, duration of 500 ps) emitted through a Koshelev antenna and monitored the consequences of electromagnetic fields exposure on the expression levels of several key genes involved in calcium metabolism, signal transduction, ROS, and energy status. We found that this treatment was mostly unable to trigger significant changes in the messenger RNA accumulation of calmodulin, Zinc-Finger protein ZAT12, NADPH oxidase/respiratory burst oxidase homolog (RBOH) isoforms D and F, Catalase (CAT2), glutamate-cystein ligase (GSH1), glutathione synthetase (GSH2), Sucrose non-fermenting-related Kinase 1 (SnRK1) and Target of rapamycin (TOR). In contrast, Ascorbate peroxidases APX-1 and APX-6 were significantly induced 3 h after the exposure. These results suggest that this treatment, although quite strong in amplitude, is mostly ineffective in inducing biological effects at the transcriptional level when delivered by an antenna. Bioelectromagnetics. 45:4–15, 2024. © 2023 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.

**Keywords:** *Arabidopsis thaliana*; Koshelev antennae; nanosecond electromagnetic pulses; gene expression; ascorbate peroxidase

## INTRODUCTION

Electromagnetic fields of various frequencies are increasingly frequent in the environment due to the fast development of wireless telecommunications and many connected objects using continuous wave (CW) or pulsed magnetic field (PMF) of high frequency within the range of 1–3 GHz. This has raised concerns and questions about their possible interactions with living cells and organisms [Cucurachi et al., 2013], including plants [Vian et al., 2016; Kaur et al., 2021]. Living tissues are indeed poor dielectrics and their interactions with electromagnetic field cause a temperature elevation

Grant sponsor: LRC BIOEM ("Laboratoire de Recherche Conventionné), grant number: LRC No. 002-2011.

Conflicts of interest: None.

*Correspondence to: Alain Vian, Univ Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, F-49000 Angers, France.
E-mail: alain.vian@univ-angers.fr

Received for review 2 September 2021; Revised 17 March 2023; Accepted 23 May 2023

DOI:10.1002/bem.22475

Published online 6 July 2023 in Wiley Online Library (wileyonlinelibrary.com).

<footnote n="1">¹Université Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Pascal, Clermont-Ferrand, France</footnote>

<footnote n="2">²Univ Angers, CHU Angers, INSERM, CNRS, MITOVASC, SFR ICAT, Angers, France</footnote>

<footnote n="3">³Univ Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, Angers, France</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.</footnote>
(thermal effect) that results from increased molecular agitation. The rate at which energy is absorbed per mass unit when cells are exposed to an electromagnetic field (expressed as W kg⁻¹) is defined as specific absorption rate (SAR). In most cases, no response has been detected in animal or human cells in response to electromagnetic fields exposure in the absence of thermal effect [Roux et al., 2011; Habauzit et al., 2014]. However, several studies have clearly suggested that biological responses occurred in plants after exposure conditions without increasing tissue temperature [Roux et al., 2006; Beaubois et al., 2007; Grémiaux et al., 2016; Kundu et al., 2021].

Aside from continuous wave (CW) or pulsed electromagnetic fields (PEMF), new medical applications [Schoenbach, 2018] and nonlethal electromagnetic weapons [Giri, 2004] use high-power nanosecond electromagnetic pulses (nsEMP). No temperature increase occurred in the EMP-treated tissue (despite their very high amplitude, up to tens of kV m⁻¹) and the resulting biological effects clearly belong to the nonthermal type [Beebe et al, 2003]. These effects may be the consequences of the intrinsic properties of these radiations, namely their very high amplitude, ultrashort duration, fast-rising front, complex temporal signature, and rich frequency spectrum. One of the main consequences of exposing cells to nsEMP is the permeabilization of the plasma membrane [Muller et al, 2001; Bai et al, 2017] resulting in several events such as increase of cytosolic calcium concentration [Vernier et al., 2003; Morotomi-Yano et al., 2012; Semenov et al., 2013]. These nsEMP (100 pulses of 10 ns at 10 Hz, 4400 kV m⁻¹) also impair mitochondrial membrane potential and microtubule growth in human glioblastoma cells [Carr et al., 2017]. These events could potentially induce signal transduction pathways that affect fundamental cell functions [Tolstykh et al., 2013]. ROS overproduction was reported after nsEMP exposure [Pakhomova et al., 2012; Nuccitelli et al., 2013; Hamada et al., 2020] and may initiate cellular transduction pathways, especially those related to calcium [Görlach et al., 2015] and changes in energy status [Morotomi-Yano et al., 2012]. In plants, exposure of *Arabidopsis* seeds or seedlings to very high amplitude nsEMP (10–100 pulses of 10–100 ns duration, 500–5000 kV m⁻¹) resulted in an increased leaf area 5–7 days after the exposure [Songnuan and Kirawanich, 2012], while longest (100 ns) treatment killed the seedlings [Eing et al., 2009]. A similar effect of nsEMF was found on the algae *Arthrospira platensis* [Buchmann et al., 2019]. At the cellular level, exposure of BY-2 tobacco cells in an electroporation cuvette (single 10 ns pulse, rise time 800 ns, 3300 kV m⁻¹) affected the cytoskeleton net, causing the nuclear

envelope disappearance and permeabilizing the plasma membrane [Berghöfer et al., 2009]. At the molecular level, significant increased transcription levels of three genes (*psy*, *crtR-b*, and *bkt 1*) involved in astaxanthin biosynthesis have been detected in the green alga *Haematococcus pluvialis* 72 h after the treatment [Bai et al., 2020]. These biological responses were obtained after very high amplitude nsEMP (0.5–5.10³ kV m⁻¹) performed through immerged electrodes in a small cuvette. In contrast, the question of the susceptibility of biological tissues to nsEMP emitted through an antenna has remained poorly studied, even if it corresponds to practical cases of exposure of humans, animals, or plants located in the immediate vicinity of sources emitting nsEMP. Here we exposed *Arabidopsis thaliana* plants to nsEMP emitted from an antenna to decipher if this treatment could evoke rapid changes in their physiology, using gene expression of key-cellular actors (calcium metabolism, ROS production/scavenging, and cell energy sensing) known to rapidly report plant perception of environmental stimuli. We record only limited change in the expression of these key genes, suggesting that nsEMP of these characteristics only trigger minor cellular responses in *A. thaliana* when they are delivered to the sample through a specialized antenna.

## MATERIALS AND METHODS

### Plant Material

Surface-sterilized seeds of *A. thaliana* ecotype Columbia were sowed in Petri dishes containing 0.5× Murashige and Skoog culture medium in 7% agar, and then placed in a controlled culture chamber (photo-synthetically active radiation of 160 µmol s⁻¹ m⁻² provided by LED tubes, photoperiod Light:Dark L:D 16:8, temperature 21 ± 1°C), until forming a rosette of approximately 1 cm (21–25 days).

### Exposure Set-Up

High-power ultrashort electromagnetic pulses plant exposures were performed in an anechoic chamber (Fig. 1). An ultrashort signal (280 ps rise-time from 10% to 90% of the amplitude, duration of 500 ps measured at 50% of the peak value) was produced using a high voltage pulse generator (Kentech model PBG5; Kentech Instruments, Wallingford, UK), monitored with a 16 GHz numerical oscilloscope (Keysight DSOV164 A-V series; Keysight, Santa Rosa, CA) and applied to a Koshelev-like ultra-wideband antenna that delivered a signal as repetitive pulses with a frequency of 50 Hz. The signal was measured with a custom-made Ddot sensor that generated a voltage proportional to the electric field derivative. Then, the temporal electric field
(thermal effect) that results from increased molecular agitation. The rate at which energy is absorbed per mass unit when cells are exposed to an electromagnetic field (expressed as W kg⁻¹) is defined as specific absorption rate (SAR). In most cases, no response has been detected in animal or human cells in response to electromagnetic fields exposure in the absence of thermal effect [Roux et al., 2011; Habauzit et al., 2014]. However, several studies have clearly suggested that biological responses occurred in plants after exposure conditions without increasing tissue temperature [Roux et al., 2006; Beaubois et al., 2007; Grémiaux et al., 2016; Kundu et al., 2021].

Aside from continuous wave (CW) or pulsed electromagnetic fields (PEMF), new medical applications [Schoenbach, 2018] and nonlethal electromagnetic weapons [Giri, 2004] use high-power nanosecond electromagnetic pulses (nsEMP). No temperature increase occurred in the EMP-treated tissue (despite their very high amplitude, up to tens of kV m⁻¹) and the resulting biological effects clearly belong to the nonthermal type [Beebe et al, 2003]. These effects may be the consequences of the intrinsic properties of these radiations, namely their very high amplitude, ultrashort duration, fast-rising front, complex temporal signature, and rich frequency spectrum. One of the main consequences of exposing cells to nsEMP is the permeabilization of the plasma membrane [Muller et al, 2001; Bai et al, 2017] resulting in several events such as increase of cytosolic calcium concentration [Vernier et al., 2003; Morotomi-Yano et al., 2012; Semenov et al., 2013]. These nsEMP (100 pulses of 10 ns at 10 Hz, 4400 kV m⁻¹) also impair mitochondrial membrane potential and microtubule growth in human glioblastoma cells [Carr et al., 2017]. These events could potentially induce signal transduction pathways that affect fundamental cell functions [Tolstykh et al., 2013]. ROS overproduction was reported after nsEMP exposure [Pakhomova et al., 2012; Nuccitelli et al., 2013; Hamada et al., 2020] and may initiate cellular transduction pathways, especially those related to calcium [Görlach et al., 2015] and changes in energy status [Morotomi-Yano et al., 2012]. In plants, exposure of *Arabidopsis* seeds or seedlings to very high amplitude nsEMP (10–100 pulses of 10–100 ns duration, 500–5000 kV m⁻¹) resulted in an increased leaf area 5–7 days after the exposure [Songnuan and Kirawanich, 2012], while longest (100 ns) treatment killed the seedlings [Eing et al., 2009]. A similar effect of nsEMF was found on the algae *Arthrospira platensis* [Buchmann et al., 2019]. At the cellular level, exposure of BY-2 tobacco cells in an electroporation cuvette (single 10 ns pulse, rise time 800 ns, 3300 kV m⁻¹) affected the cytoskeleton net, causing the nuclear

envelope disappearance and permeabilizing the plasma membrane [Berghöfer et al., 2009]. At the molecular level, significant increased transcription levels of three genes (*psy*, *crtR-b*, and *bkt 1*) involved in astaxanthin biosynthesis have been detected in the green alga *Haematococcus pluvialis* 72 h after the treatment [Bai et al., 2020]. These biological responses were obtained after very high amplitude nsEMP (0.5–5.10³ kV m⁻¹) performed through immerged electrodes in a small cuvette. In contrast, the question of the susceptibility of biological tissues to nsEMP emitted through an antenna has remained poorly studied, even if it corresponds to practical cases of exposure of humans, animals, or plants located in the immediate vicinity of sources emitting nsEMP. Here we exposed *Arabidopsis thaliana* plants to nsEMP emitted from an antenna to decipher if this treatment could evoke rapid changes in their physiology, using gene expression of key-cellular actors (calcium metabolism, ROS production/scavenging, and cell energy sensing) known to rapidly report plant perception of environmental stimuli. We record only limited change in the expression of these key genes, suggesting that nsEMP of these characteristics only trigger minor cellular responses in *A. thaliana* when they are delivered to the sample through a specialized antenna.

## MATERIALS AND METHODS

### Plant Material

Surface-sterilized seeds of *A. thaliana* ecotype Columbia were sowed in Petri dishes containing 0.5× Murashige and Skoog culture medium in 7% agar, and then placed in a controlled culture chamber (photo-synthetically active radiation of 160 µmol s⁻¹ m⁻² provided by LED tubes, photoperiod Light:Dark L:D 16:8, temperature 21 ± 1°C), until forming a rosette of approximately 1 cm (21–25 days).

### Exposure Set-Up

High-power ultrashort electromagnetic pulses plant exposures were performed in an anechoic chamber (Fig. 1). An ultrashort signal (280 ps rise-time from 10% to 90% of the amplitude, duration of 500 ps measured at 50% of the peak value) was produced using a high voltage pulse generator (Kentech model PBG5; Kentech Instruments, Wallingford, UK), monitored with a 16 GHz numerical oscilloscope (Keysight DSOV164 A-V series; Keysight, Santa Rosa, CA) and applied to a Koshelev-like ultra-wideband antenna that delivered a signal as repetitive pulses with a frequency of 50 Hz. The signal was measured with a custom-made Ddot sensor that generated a voltage proportional to the electric field derivative. Then, the temporal electric field
6 Porcher et al.

<figure><img src="image_2.png" /><figcaption>Fig. 1. Experimental design and exposure characteristics. (a) Left: schematic diagram of the exposure apparatus. The signal is generated using an Agilent 8110A pulse generator, triggering a shielded Kentech model PBG5 high voltage solid state pulser (d) and then injected to a Koshelev-like antenna (b). The plant samples were placed in the antenna (e). The Koshelev-like antenna and the Kentech pulser were both placed inside an anechoic chamber (a). Right: photograph showing the installation of the set-up inside the anechoic chamber (a), the Koshelev antenna (b) and the sparkler (c), and the Kentech model PBG5 high voltage solid state pulser (d). (b) Field amplitude time evolution (left) and the signal spectrum transmitted by the Koshelev antenna (right) in response to the excitation provided by the PBG5 solid-state pulser at exposed plant location.</figcaption></figure>

$E(t)$ was obtained from the measured voltage $v(t')$ using Equation (1):

$$E(t) = \frac{1}{R \cdot A_{eff} \cdot \varepsilon_0} \int V(t') dt' \quad (1)$$

where R is the sensor impedance (50 Ω), $A_{eff}$ the effective cross-section of the Ddot, and $\varepsilon_0$ the dielectric permittivity of vacuum. For technical reasons, this nonintrusive sensor was fixed 100 mm above the antenna base, where the gap between the plates of the antenna was $g_d = 26$ mm. The maximum field amplitude deduced at this point was $E_{measured} = 753$ kV m⁻¹. The plants were positioned 300 mm above the antenna base, where the distance between the plates was 82.5 mm. Consequently, the electric field amplitude at this point was 237 kV m⁻¹.

We exposed plants to 30,000 electromagnetic pulses (about 10 min) by placing the samples 300 mm above the antenna base (Fig. 1a). We measured the temporal electric field at the exposure location in the absence of the Petri dish (Fig. 1b). From there, numerical simulations were performed to estimate the induced electric field and the SAR inside the Petri dish. We imported the temporal profile in the commercial simulation software CST Studio Suite 2019 to generate an incident plane wave on Petri dish containing 0.5× Murashige and Skoog culture medium in 7% agar. Perfectly matched layer (PML) conditions at the boundaries of the computational domain allow us to simulate open space. We used adaptive mesh refinement associated with extra space for the application of the PML leading to 515,871 mesh cells to get accurate
6 Porcher et al.

<figure><img src="image_2.png" /><figcaption>Fig. 1. Experimental design and exposure characteristics. (a) Left: schematic diagram of the exposure apparatus. The signal is generated using an Agilent 8110A pulse generator, triggering a shielded Kentech model PBG5 high voltage solid state pulser (d) and then injected to a Koshelev-like antenna (b). The plant samples were placed in the antenna (e). The Koshelev-like antenna and the Kentech pulser were both placed inside an anechoic chamber (a). Right: photograph showing the installation of the set-up inside the anechoic chamber (a), the Koshelev antenna (b) and the sparkler (c), and the Kentech model PBG5 high voltage solid state pulser (d). (b) Field amplitude time evolution (left) and the signal spectrum transmitted by the Koshelev antenna (right) in response to the excitation provided by the PBG5 solid-state pulser at exposed plant location.</figcaption></figure>

$E(t)$ was obtained from the measured voltage $v(t')$ using Equation (1):

$$E(t) = \frac{1}{R \cdot A_{eff} \cdot \varepsilon_0} \int V(t') dt' \quad (1)$$

where $R$ is the sensor impedance (50 Ω), $A_{eff}$ the effective cross-section of the Ddot, and $\varepsilon_0$ the dielectric permittivity of vacuum. For technical reasons, this nonintrusive sensor was fixed 100 mm above the antenna base, where the gap between the plates of the antenna was $g_d = 26$ mm. The maximum field amplitude deduced at this point was $E_{measured} = 753$ kV m⁻¹. The plants were positioned 300 mm above the antenna base, where the distance between the plates was 82.5 mm. Consequently, the electric field amplitude at this point was 237 kV m⁻¹.

We exposed plants to 30,000 electromagnetic pulses (about 10 min) by placing the samples 300 mm above the antenna base (Fig. 1a). We measured the temporal electric field at the exposure location in the absence of the Petri dish (Fig. 1b). From there, numerical simulations were performed to estimate the induced electric field and the SAR inside the Petri dish. We imported the temporal profile in the commercial simulation software CST Studio Suite 2019 to generate an incident plane wave on Petri dish containing 0.5× Murashige and Skoog culture medium in 7% agar. Perfectly matched layer (PML) conditions at the boundaries of the computational domain allow us to simulate open space. We used adaptive mesh refinement associated with extra space for the application of the PML leading to 515,871 mesh cells to get accurate
CST simulation results. The maximum time domain solver duration was driven through a total solver duration in second rather than an energy criterion (accuracy was set to “No Check” for solver settings). We wanted to ensure all signal fall back to zero for accurate Fast Fourier Transforms and SAR results. The dielectric parameters of this medium were experimentally measured using an accurate transmission technique (IndexSAR Diline, Horsham, UK), associated with the conductivity model as shown in the following Equation (2):

$$ \varepsilon(\omega) = \varepsilon_r(\omega) - j \frac{\sigma(\omega)}{\omega\varepsilon_0} \quad (2) $$

where $εr$ corresponds to the real part of the dispersive model, $σ$ in S m⁻¹ is the electric conductivity and $ω$ is the angular frequency in rad s⁻¹. The experimental values obtained for $εr$ and for $σ$ were fitted to be used as CST new materials to obtain the time evolution of the electric field (Fig. 2a) and the frequency spectrum (Fig. 2b) in two points of the Petri dish loaded with culture medium (central and peripheral, 5 mm from the edge).

Arabidopsis plants (1 cm rosette stage, 3 weeks old) were placed under an illumination ramp (150 µmol s⁻¹ m⁻²) in the immediate vicinity of the anechoic chamber, moved into the Koshelev antenna and subjected to 30,000 nsEMP (for a total duration of about 10 min in the absence of light) and then immediately returned under the illumination ramp until their harvest. Plants were collected before the exposure (T0) and 15, 30, 60, and 180 min after the end of the exposure and immediately

frozen in dry ice. Control plants were manipulated the same way and harvested similarly at the same time points, except that they were not subjected to nsEMP exposure.

RNA Isolation and Complementary DNA (cDNA) Synthesis

RNA was isolated from the whole aerial part of the exposed and nonexposed plants using the RNeasy Plant Mini Kit including DNase treatment (Qiagen, Courtaboeuf, France), according to the manufacturer instructions. Briefly, plants (30–50 mg) were grounded to a fine powder in a Bertin tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France), resuspended in lysis buffer and cleaned on a QIAshredder spin column (Qiagen). After ethanol addition (50% of the volume), and purification step on a RNeasy spin column, possible contamination with genomic DNA was removed with a DNase treatment (Qiagen), and the RNA was eluted in 30 µl of RNase-free water. RNA samples were quantified using Nanodrop (Thermo Scientific, Villebon sur Yvette, France), and 500 ng of total RNA was used to prime cDNA synthesis using the Superscript II first Strand synthesis system for real-time quantitative polymerase chain reaction (RT-PCR) following the manufacturer recommendations (Invitrogen, Villebon sur Yvette, France).

Quantification of Gene Expression

Relative gene expression was obtained using RT-qPCR. Amplifications were performed in white PCR

<figure><img src="image_3.png" /><figcaption>Fig. 2. Evaluation of field characteristics in the Petri dish. Simulations were performed using CST-studio 2019 software with experimentally determined values of permittivity and conductivity of the medium present in the Petri dish. (a) Time evolution of the electric field in the center (continuous line) and periphery (dotted line) of the Petri dish. (b) Spectrum distribution of the electric field in the center (continuous line) and periphery (dotted line) of the Petri dish.</figcaption></figure>
CST simulation results. The maximum time domain solver duration was driven through a total solver duration in second rather than an energy criterion (accuracy was set to “No Check” for solver settings). We wanted to ensure all signal fall back to zero for accurate Fast Fourier Transforms and SAR results. The dielectric parameters of this medium were experimentally measured using an accurate transmission technique (IndexSAR Diline, Horsham, UK), associated with the conductivity model as shown in the following Equation (2):

$$ \varepsilon(\omega) = \varepsilon_r(\omega) - j \frac{\sigma(\omega)}{\omega\varepsilon_0} \quad (2) $$

where $\varepsilon r$ corresponds to the real part of the dispersive model, $\sigma$ in S m⁻¹ is the electric conductivity and $\omega$ is the angular frequency in rad s⁻¹. The experimental values obtained for $\varepsilon r$ and for $\sigma$ were fitted to be used as CST new materials to obtain the time evolution of the electric field (Fig. 2a) and the frequency spectrum (Fig. 2b) in two points of the Petri dish loaded with culture medium (central and peripheral, 5 mm from the edge).

Arabidopsis plants (1 cm rosette stage, 3 weeks old) were placed under an illumination ramp (150 µmol s⁻¹ m⁻²) in the immediate vicinity of the anechoic chamber, moved into the Koshelev antenna and subjected to 30,000 nsEMP (for a total duration of about 10 min in the absence of light) and then immediately returned under the illumination ramp until their harvest. Plants were collected before the exposure (T0) and 15, 30, 60, and 180 min after the end of the exposure and immediately

frozen in dry ice. Control plants were manipulated the same way and harvested similarly at the same time points, except that they were not subjected to nsEMP exposure.

RNA Isolation and Complementary DNA (cDNA) Synthesis

RNA was isolated from the whole aerial part of the exposed and nonexposed plants using the RNeasy Plant Mini Kit including DNAse treatment (Qiagen, Courtaboeuf, France), according to the manufacturer instructions. Briefly, plants (30–50 mg) were grounded to a fine powder in a Bertin tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France), resuspended in lysis buffer and cleaned on a QIAshredder spin column (Qiagen). After ethanol addition (50% of the volume), and purification step on a RNeasy spin column, possible contamination with genomic DNA was removed with a DNAse treatment (Qiagen), and the RNA was eluted in 30 µl of RNase-free water. RNA samples were quantified using Nanodrop (Thermo Scientific, Villebon sur Yvette, France), and 500 ng of total RNA was used to prime cDNA synthesis using the Superscript II first Strand synthesis system for real-time quantitative polymerase chain reaction (RT-PCR) following the manufacturer recommendations (Invitrogen, Villebon sur Yvette, France).

Quantification of Gene Expression

Relative gene expression was obtained using RT-qPCR. Amplifications were performed in white PCR

<figure><img src="image_3.png" /><figcaption>Fig. 2. Evaluation of field characteristics in the Petri dish. Simulations were performed using CST-studio 2019 software with experimentally determined values of permittivity and conductivity of the medium present in the Petri dish. (a) Time evolution of the electric field in the center (continuous line) and periphery (dotted line) of the Petri dish. (b) Spectrum distribution of the electric field in the center (continuous line) and periphery (dotted line) of the Petri dish.</figcaption></figure>
plates (Bio-Rad, Marne-la-Coquette, France) in a final volume of 10 µl of RT-qPCR reaction mix containing 0.6 µl primers pairs (10 µM), 3 µl iQ SYBR Green supermix (Bio-Rad), 4.4 µl of ultrapure water and completed with 2 µl of 50 times diluted cDNA, using the following amplification program: 95°C for 1 min (enzyme initiating step), 40 cycles with 5 s at 95°C following by 30 s at 60°C and a final melting step achieving an increase of 0.5°C every 2 s from 60°C to 95°C. Fluorescence of each well was detected at the end of each PCR cycle using the CFX384 Real-Time System (Bio-Rad). Relative transcript abundance was expressed to the control condition after $2^{-\Delta\Delta C_t}$ calculation, as described by Livak and Schmittgen (2001). The following primers (5'-3') were used to amplify eleven candidate genes and two house-keeping genes (*ACTIN* and *glyceraldehyde-3-phosphate dehydrogenase*, *GAPDH*): *ACTIN* (Forward: GCCATCCAAGCTGTTCTCTC and Reverse: CCCTCGTAGATTGGCACAGT), *GAPDH* (Forward: TCAGGAACCCTGAGGACATC and Reverse CGTTGACACCAACAACGAAC), *ZAT12* (Forward: TGCGAGTCACAAGAAGCCTA and Reverse: GTGTCCTCCCAAAGCTTGTC), *RBOHD* (Forward: CGTGGAGATCACGCTAGACA and Reverse: AGGA GGTGGTGTTGTTGAGG), *RBOHF* (Forward: GGTGC ACCAGCACAAGATTA and Reverse: TGAAATCC GAGATCGAATCC), *CAT2* (Forward: CGAGGTAT GACCAGGTTCGT and Reverse: CTCCAGGCTCCTT GAAGTTG), *APX1* (Forward: GCACTATTGGAC GACCCTGT and Reverse: AGCAAACCCAAGCTCA GAAA), *APX6* (Forward: CGGTGCGACTCGTTTT CATC and Reverse: GCTTGCCTTCTGGATCAGGT), *GSH1* (Forward: CGCTCTTGTCTCAAGCAGGA and Reverse: CTAAGCTCGAACTGACCCCC), *GSH2* (Forward: CGTTTCCTGGCCTTAGTCGT and Reverse: CCAAAGCCCAGCAAAGCATT), *SnRK1* (Forward: GCTGAGCATGCATTGACAGG and Reverse: CAC ACCACAGCTCCAGACAT) and *TOR* (Forward: AGGTGTGGCAAGCACTTTTG and Reverse: TTCGA CAAAGCGAGGCAAAC). *TCH1* (Forward: ATG GAAACGGCACTATCGAC and Reverse ATTGGT CATCACATGGCGTA) [Braam and Davis, 1990] was selected over other calmodulin genes since they had proven to respond to a wide variety of environmental stimuli including exposure to electromagnetic fields [Roux et al., 2006; Rammal et al., 2014].

## Statistical Analysis

Statistical analysis was performed with the Rstudio statistical software using statistical tests as described in figure captions. Graphics were produced using Veusz 3.4 plotting software.

## RESULTS

### Characteristics of the Electromagnetic Pulses

The time-dependent electric field emitted by the antenna (Fig. 1b, left) showed the presence of a major peak displaying a very short 280 ps rise-time measured from 10% to 90% of the amplitude, a duration of 500 ps measured at 50% of the peak value (237 kV m⁻¹) and a repetition rate of 50 Hz (30,000 pulses in 10 min). After a Fast Fourier Transform (FFT) operation, the frequency spectrum (Fig. 1b, right) showed that the electromagnetic pulses are mainly constituted by frequencies lower than 1.5 GHz (where the signal reaches -20 dB). The contribution of higher frequencies decreasing rapidly (achieving -40 dB at 2.5 GHz), becoming then negligible. Thus, the signal appears as 30,000 high amplitude (237 kV m⁻¹) and short duration sharp pulses emitted by the Koshelev antenna and mainly composed of frequencies below 2 GHz.

### Field Distribution and SAR Levels in the Petri Dish

The characteristics of electromagnetic pulses were evaluated in the Petri dish using CST-studio software and a custom model of the Petri dish. The temporal evolution of the electric field showed in the center of the Petri dish (Fig. 2a, solid line) a major peak displaying a short rise-time, while at the edges of the Petri Dish (Fig. 2a, dotted line), the signal appeared more complex, with the presence of multiple peaks. The spectral distribution of the electric field was quite similar to that measured in the antenna, at the center of the Petri dish as well as near the edges (Fig. 2b, solid and dotted lines, respectively), showing that the signal was mainly constituted of frequencies below 2 GHz. The resulting electromagnetic field distributions were simulated for different frequencies (400, 1500, 2000, and 2500 MHz) in the Petri dish using CST-studio (Fig. 3a–d). The field amplitude appeared to be very low, under $2 \times 10^{-5} \text{ V m}^{-1}$ at 400 MHz, peaking at about $5.57 \times 10^{-5} \text{ V m}^{-1}$ at 1500 MHz (Fig. 3b) and decreased under $2 \times 10^{-5} \text{ V m}^{-1}$ at 2000 and 2500 MHz (Fig. 3c and d). While the distribution was quite uniform at 400 MHz (Fig. 3a), some heterogeneity appeared at higher frequencies, displaying hot spots whose number increased with the frequency, improving in turn the homogeneity of the field (Fig. 3b–d). Because of the low field amplitudes, the SAR levels estimated in the Petri dish were extremely low (Fig. 3e–h), with values in the range of $10^{-13}$ to $10^{-16} \text{ W kg}^{-1}$, the minimum values occurring at 400 MHz and the highest ones at
plates (Bio-Rad, Marne-la-Coquette, France) in a final volume of 10 µl of RT-qPCR reaction mix containing 0.6 µl primers pairs (10 µM), 3 µl iQ SYBR Green supermix (Bio-Rad), 4.4 µl of ultrapure water and completed with 2 µl of 50 times diluted cDNA, using the following amplification program: 95°C for 1 min (enzyme initiating step), 40 cycles with 5 s at 95°C following by 30 s at 60°C and a final melting step achieving an increase of 0.5°C every 2 s from 60°C to 95°C. Fluorescence of each well was detected at the end of each PCR cycle using the CFX384 Real-Time System (Bio-Rad). Relative transcript abundance was expressed to the control condition after $2^{-\Delta\Delta C_t}$ calculation, as described by Livak and Schmittgen (2001). The following primers (5'-3') were used to amplify eleven candidate genes and two house-keeping genes (*ACTIN* and *glyceraldehyde-3-phosphate dehydrogenase*, *GAPDH*): *ACTIN* (Forward: GCCATCCAAGCTGTTCTCTC and Reverse: CCCTCGTAGATTGGCACAGT), *GAPDH* (Forward: TCAGGAACCCTGAGGACATC and Reverse CGTTGACACCAACAACGAAC), *ZAT12* (Forward: TGCGAGTCACAAGAAGCCTA and Reverse: GTGTCCTCCCAAAGCTTGTC), *RBOHD* (Forward: CGTGGAGATCACGCTAGACA and Reverse: AGGA GGTGGTGTTGTTGAGG), *RBOHF* (Forward: GGTGC ACCAGCACAAGATTA and Reverse: TGAAATCC GAGATCGAATCC), *CAT2* (Forward: CGAGGTAT GACCAGGTTCGT and Reverse: CTCCAGGCTCCTT GAAGTTG), *APX1* (Forward: GCACTATTGGAC GACCCTGT and Reverse: AGCAAACCCAAGCTCA GAAA), *APX6* (Forward: CGGTGCGACTCGTTTT CATC and Reverse: GCTTGCCTTCTGGATCAGGT), *GSH1* (Forward: CGCTCTTGTCTCAAGCAGGA and Reverse: CTAAGCTCGAACTGACCCCC), *GSH2* (Forward: CGTTTCCTGGCCTTAGTCGT and Reverse: CCAAAGCCCAGCAAAGCATT), *SnRK1* (Forward: GCTGAGCATGCATTGACAGG and Reverse: CAC ACCACAGCTCCAGACAT) and *TOR* (Forward: AGGTGTGGCAAGCACTTTTG and Reverse: TTCGA CAAAGCGAGGCAAAC). *TCH1* (Forward: ATG GAAACGGCACTATCGAC and Reverse ATTGGT CATCACATGGCGTA) [Braam and Davis, 1990] was selected over other calmodulin genes since they had proven to respond to a wide variety of environmental stimuli including exposure to electromagnetic fields [Roux et al., 2006; Rammal et al., 2014].

## Statistical Analysis

Statistical analysis was performed with the Rstudio statistical software using statistical tests as described in figure captions. Graphics were produced using Veusz 3.4 plotting software.

## RESULTS

### Characteristics of the Electromagnetic Pulses

The time-dependent electric field emitted by the antenna (Fig. 1b, left) showed the presence of a major peak displaying a very short 280 ps rise-time measured from 10% to 90% of the amplitude, a duration of 500 ps measured at 50% of the peak value (237 kV m⁻¹) and a repetition rate of 50 Hz (30,000 pulses in 10 min). After a Fast Fourier Transform (FFT) operation, the frequency spectrum (Fig. 1b, right) showed that the electromagnetic pulses are mainly constituted by frequencies lower than 1.5 GHz (where the signal reaches -20 dB). The contribution of higher frequencies decreasing rapidly (achieving -40 dB at 2.5 GHz), becoming then negligible. Thus, the signal appears as 30,000 high amplitude (237 kV m⁻¹) and short duration sharp pulses emitted by the Koshelev antenna and mainly composed of frequencies below 2 GHz.

### Field Distribution and SAR Levels in the Petri Dish

The characteristics of electromagnetic pulses were evaluated in the Petri dish using CST-studio software and a custom model of the Petri dish. The temporal evolution of the electric field showed in the center of the Petri dish (Fig. 2a, solid line) a major peak displaying a short rise-time, while at the edges of the Petri Dish (Fig. 2a, dotted line), the signal appeared more complex, with the presence of multiple peaks. The spectral distribution of the electric field was quite similar to that measured in the antenna, at the center of the Petri dish as well as near the edges (Fig. 2b, solid and dotted lines, respectively), showing that the signal was mainly constituted of frequencies below 2 GHz. The resulting electromagnetic field distributions were simulated for different frequencies (400, 1500, 2000, and 2500 MHz) in the Petri dish using CST-studio (Fig. 3a–d). The field amplitude appeared to be very low, under $2 \times 10^{-5} \text{ V m}^{-1}$ at 400 MHz, peaking at about $5.57 \times 10^{-5} \text{ V m}^{-1}$ at 1500 MHz (Fig. 3b) and decreased under $2 \times 10^{-5} \text{ V m}^{-1}$ at 2000 and 2500 MHz (Fig. 3c and d). While the distribution was quite uniform at 400 MHz (Fig. 3a), some heterogeneity appeared at higher frequencies, displaying hot spots whose number increased with the frequency, improving in turn the homogeneity of the field (Fig. 3b–d). Because of the low field amplitudes, the SAR levels estimated in the Petri dish were extremely low (Fig. 3e–h), with values in the range of $10^{-13}$ to $10^{-16} \text{ W kg}^{-1}$, the minimum values occurring at 400 MHz and the highest ones at
<figure><img src="image_4.png" /><figcaption>Fig. 3. Field distribution and specific absorption rate (SAR) values in the Petri dish. Field distribution and SAR value were determined using CST-studio 2019 software and a custom-made model of Petri dish. (a–d) Field distribution inside the Petri dish for different frequencies: 400 (a), 1500 (b), 2000 (c), and 2500 (D) MHz. The calculated values are rendered according to the color scale on the right. (e, f) SAR levels inside the Petri dish for different frequencies: 400 (e), 1500 (f), 2000 (g), and 2500 (h) MHz. The calculated values are rendered according to the color scale on the right.</figcaption></figure>

1500 MHz. The SAR was generally higher in the central part of the Petri dish (Fig. 3e–g), except at 2500 MHz where it maximized in the intermediate zone of the Petri dish (Fig. 3h). These very low SAR values confirm that plant exposure to nsEMP is athermal in nature and does not cause tissue heating.

## Changes in Gene Expression

The gene expression level encoding for calmodulin, involved in calcium signaling, which is an extremely sensitive marker of the perception of an environmental stimulus in plants [Braam and Davis, 1990], remains almost constant in the control and exposed plants all along the kinetics (Fig. 4a),

only displaying a significant lower abundance 30 min after the exposure (0.05 < *P* < 0.10). Similarly, the messenger RNA (mRNA) abundance of ZAT12 transcription factor, a universal marker of environmental stress in plants, remains similar in the control, unexposed plants compared to nsEMP exposed samples (Fig. 4b) all along the kinetics.

Gene expression of two isoforms of the NADPH oxidases/respiratory burst oxidase homologs (*RBOH-D* and *-F*, Fig. 5a), one of the major actors of the main pathway for H₂O₂ production in plants were analyzed, showing no change (*P* < 0.05) over the 180 min of the kinetics in control and exposed plants. A significant lower abundance of *RBOH-F* transcript was however noticed 30 min after the exposure (0.05 < *P* < 0.10).
<figure><img src="image_4.png" /><figcaption>Fig. 3. Field distribution and specific absorption rate (SAR) values in the Petri dish. Field distribution and SAR value were determined using CST-studio 2019 software and a custom-made model of Petri dish. (a–d) Field distribution inside the Petri dish for different frequencies: 400 (a), 1500 (b), 2000 (c), and 2500 (D) MHz. The calculated values are rendered according to the color scale on the right. (e, f) SAR levels inside the Petri dish for different frequencies: 400 (e), 1500 (f), 2000 (g), and 2500 (h) MHz. The calculated values are rendered according to the color scale on the right.</figcaption></figure>

1500 MHz. The SAR was generally higher in the central part of the Petri dish (Fig. 3e–g), except at 2500 MHz where it maximized in the intermediate zone of the Petri dish (Fig. 3h). These very low SAR values confirm that plant exposure to nsEMP is athermal in nature and does not cause tissue heating.

## Changes in Gene Expression

The gene expression level encoding for calmodulin, involved in calcium signaling, which is an extremely sensitive marker of the perception of an environmental stimulus in plants [Braam and Davis, 1990], remains almost constant in the control and exposed plants all along the kinetics (Fig. 4a),

only displaying a significant lower abundance 30 min after the exposure (0.05 < *P* < 0.10). Similarly, the messenger RNA (mRNA) abundance of ZAT12 transcription factor, a universal marker of environmental stress in plants, remains similar in the control, unexposed plants compared to nsEMP exposed samples (Fig. 4b) all along the kinetics.

Gene expression of two isoforms of the NADPH oxidases/respiratory burst oxidase homologs (*RBOH-D* and *-F*, Fig. 5a), one of the major actors of the main pathway for H₂O₂ production in plants were analyzed, showing no change (*P* < 0.05) over the 180 min of the kinetics in control and exposed plants. A significant lower abundance of *RBOH-F* transcript was however noticed 30 min after the exposure (0.05 < *P* < 0.10).
<figure><img src="image_5.png" /></figure>

Fig. 4. Effects of nanosecond electromagnetic pulses (nsEMP) on signal-transduction genes expression. Relative transcript accumulations of (a) the calmodulin (TCH-1) gene and (b) the zinc finger protein (ZAT12) gene. Data are means of three independent biological replicates ± SE (each comprising two technical replicates) of relative expression compared to T0 after normalization using expression of reference genes (Actin and GAPDH). The dot indicates a difference (0.05 < *P* < 0.10) between unexposed (control) and ns-EMP exposed plants after a nonparametric Wilcoxon rank sum test.

<figure><img src="image_6.png" /></figure>

Fig. 5. Effects of nanosecond electromagnetic pulses (nsEMP) on the expression of genes involved in H₂O₂ production and scavenging. Relative transcript accumulations of (a) the isoforms D and F of the NADPH oxidase/respiratory burst oxidase homologs (RBOH-D and -F) and (b) the catalase 2 (CAT2) genes. Data are means of three biological independent replicates ± SE (each comprising two technical repeats) of relative expression compared to T0 after normalization using expression of reference genes (Actin and GAPDH). The dot indicates a difference (0.05 < *P* < 0.10) between unexposed (control) and ns-EMP exposed plants after a nonparametric Wilcoxon rank sum test.

The gene expression of *CAT2* catalase was also unchanged in controls and nsEMP-exposed plants, suggesting that no burst of H₂O₂ occurred after plant exposure (Fig. 5b), even if some variations of limited amplitude (a slight diminution 30–60 min after the beginning of the experiment) were noted, both in control and exposed plants.

While catalase is rather involved in the rapid detoxification of large amounts of hydrogen peroxide, it seemed interesting to explore the changes that exposure of plants to ns-EMP might induce in the gene expression levels of enzymes of the ascorbic acid/glutathione cycle of H₂O₂ scavenging, involved in the fine-tuning of H₂O₂ detoxification. Ascorbic acid
<figure><img src="image_5.png" /></figure>

Fig. 4. Effects of nanosecond electromagnetic pulses (nsEMP) on signal-transduction genes expression. Relative transcript accumulations of (a) the calmodulin (TCH-1) gene and (b) the zinc finger protein (ZAT12) gene. Data are means of three independent biological replicates ± SE (each comprising two technical replicates) of relative expression compared to T0 after normalization using expression of reference genes (Actin and GAPDH). The dot indicates a difference (0.05 < *P* < 0.10) between unexposed (control) and ns-EMP exposed plants after a nonparametric Wilcoxon rank sum test.

<figure><img src="image_6.png" /></figure>

Fig. 5. Effects of nanosecond electromagnetic pulses (nsEMP) on the expression of genes involved in H₂O₂ production and scavenging. Relative transcript accumulations of (a) the isoforms D and F of the NADPH oxidase/respiratory burst oxidase homologs (RBOH-D and -F) and (b) the catalase 2 (CAT2) genes. Data are means of three biological independent replicates ± SE (each comprising two technical repeats) of relative expression compared to T0 after normalization using expression of reference genes (Actin and GAPDH). The dot indicates a difference (0.05 < *P* < 0.10) between unexposed (control) and ns-EMP exposed plants after a nonparametric Wilcoxon rank sum test.

The gene expression of *CAT2* catalase was also unchanged in controls and nsEMP-exposed plants, suggesting that no burst of H₂O₂ occurred after plant exposure (Fig. 5b), even if some variations of limited amplitude (a slight diminution 30–60 min after the beginning of the experiment) were noted, both in control and exposed plants.

While catalase is rather involved in the rapid detoxification of large amounts of hydrogen peroxide, it seemed interesting to explore the changes that exposure of plants to ns-EMP might induce in the gene expression levels of enzymes of the ascorbic acid/glutathione cycle of H₂O₂ scavenging, involved in the fine-tuning of H₂O₂ detoxification. Ascorbic acid
peroxidases (APX-1 and APX-6) gene expression was essentially similar in control and exposed plants and both were very constant along the kinetics (Fig. 6a and b), except 180 min after the exposure where both APX-1 and APX-6 gene expression were significantly higher (2.5- and 1.2-fold, respectively). No significant changes were noticed in the glutamate-cystein ligase (GSH1) and GSH synthetase (GSH2) gene expression patterns, that were both very constant along the kinetics in both control and exposed samples (Fig. 6c and d).

Here we monitored the transcript abundance of the two kinases (SnRK1 and TOR) that

antagonistically react to the energy status of the cells which is affected in plants due to the perception of environmental signals. Here we observed that *SnRK1* and *TOR* expression remained globally low and constant, not being enhanced after exposing plants to nsEMP (Fig. 7a). Only TOR expression level showed a significant change (0.05 *< P *< 0.10) 30 min after the exposure. The expression level of TOR appears more irregular with a higher—however not statistically significant —expression level noticed in exposed plants 60 min after the treatment.

<figure><img src="image_7.png" /></figure>

Fig. 6. Effects of nanosecond electromagnetic pulses (nsEMP) on the expression of genes involved in H₂O₂ scavenging through Ascorbic acid-glutathione cycle. Relative gene expression of (a, b) isoforms 1 and 6 of the ascorbate peroxidases (APX1 and APX6), (c) the glutamate-cystein ligase (GSH1) and (d) the glutathione synthetase (GSH2) genes. Data are means of three biological independent replicates ± SE (each comprising two technical repeats) of relative expression compared to T0 after normalization using expression of reference genes (Actin and GAPDH). Significant differences (*P* < 0.05) between unexposed (control) and ns-EMP exposed plants are indicated by an asterisk (*) after nonparametric Wilcoxon rank sum test.
peroxidases (APX-1 and APX-6) gene expression was essentially similar in control and exposed plants and both were very constant along the kinetics (Fig. 6a and b), except 180 min after the exposure where both APX-1 and APX-6 gene expression were significantly higher (2.5- and 1.2-fold, respectively). No significant changes were noticed in the glutamate-cystein ligase (GSH1) and GSH synthetase (GSH2) gene expression patterns, that were both very constant along the kinetics in both control and exposed samples (Fig. 6c and d).

Here we monitored the transcript abundance of the two kinases (SnRK1 and TOR) that

antagonistically react to the energy status of the cells which is affected in plants due to the perception of environmental signals. Here we observed that *SnRK1* and *TOR* expression remained globally low and constant, not being enhanced after exposing plants to nsEMP (Fig. 7a). Only TOR expression level showed a significant change (0.05 *< P *< 0.10) 30 min after the exposure. The expression level of TOR appears more irregular with a higher—however not statistically significant —expression level noticed in exposed plants 60 min after the treatment.

<figure><img src="image_7.png" /></figure>

Fig. 6. Effects of nanosecond electromagnetic pulses (nsEMP) on the expression of genes involved in H₂O₂ scavenging through Ascorbic acid-glutathione cycle. Relative gene expression of (a, b) isoforms 1 and 6 of the ascorbate peroxidases (APX1 and APX6), (c) the glutamate-cystein ligase (GSH1) and (d) the glutathione synthetase (GSH2) genes. Data are means of three biological independent replicates ± SE (each comprising two technical repeats) of relative expression compared to T0 after normalization using expression of reference genes (Actin and GAPDH). Significant differences (*P* < 0.05) between unexposed (control) and ns-EMP exposed plants are indicated by an asterisk (*) after nonparametric Wilcoxon rank sum test.
<figure><img src="image_8.png" /><figcaption>Fig. 7. Effects of nanosecond electromagnetic pulses (nsEMP) on gene expression involved in cell energy sensing. Relative gene expression of (a) *SnRK1* (Sucrose nonfermenting-related kinase 1) and (b) the *TOR* (target of rapamycin) genes. Data are means of three biological independent replicates ± SE (each comprising two technical repeats) of relative expression compared to T0 after normalization using expression of reference genes (*Actin* and *GAPDH*). The dot indicates a difference (0.05 < *P* < 0.10) between unexposed (control) and ns-EMP exposed plants after a nonparametric Wilcoxon rank sum test.</figcaption></figure>

DISCUSSION

The objective of this study was to determine if
high-amplitude ultrashort electromagnetic field
pulses emitted from an antenna could induce changes
in the expression of key genes involved in the early
stages of perception of environmental stress. We set-
up an experimental system that allowed us to expose
the plants in the open volume of an anechoic
chamber, which is the condition ensuring the
minimum plant handling to avoid possible interaction
with the plant responses to nsEMP exposure. It is,
also, much closer to the situations that are encoun-
tered in civilian or military applications, where
nsEMP are emitted in the environment. The nsEMP
emitted from an antenna are, indeed, much less
commonly used than their applications through
immersed electrodes in direct contact with the
culture medium in a small, dedicated cuvette. This
last kind of equipment has the advantage of allowing
the generation of very high amplitude electromag-
netic pulses (up to several hundreds of V m⁻¹) with
rise times of a few ns to several ms [Schunck
et al., 2014], but restricts the application to cell
cultures or small biological samples. Using this kind
of equipment led to achieve various biological
responses in animal and human cells, including
membrane permeabilization [Joshi and Schoen-
bach, 2010], lower mitochondrial membrane poten-
tial [Beebe et al., 2012], cytosolic calcium increase
[Vernier et al., 2003; Semenov et al., 2013, 2015],

ROS production [Nuccitelli et al., 2013] and altera-
tion in gene expression [Zhang et al., 2014; Roth
et al, 2016].

Here we used a specialized (Koshelev) antenna
[Godard et al., 2010] that was able to deliver the high
amplitude complex nanosecond pulses. The plants
were placed along the central axis of the antenna, a
configuration shown to maximize the energy absorp-
tion [Ji et al., 2006]. Using this set-up (Fig. 1a), we
were able to expose plants to high amplitude
(237 kV m⁻¹) short duration (500 ps) EMPs that
display a very short raising time (about 280 ps),
values within the orders of magnitude for this type of
electromagnetic pulses [Schunck et al., 2014]. The
frequency distribution of the nsEMP used in the
present work (Fig. 1b) showed that it mainly accounts
for frequencies much lower than 2 GHz.

Simulation work performed with CST-Studio
software using dielectric permittivity and electrical
conductivity determined with a Diline equipment
showed that the resulting field inside the Petri dish
had a temporal signature similar to that measured in
the antenna in the center of the Petri dish (Fig. 2a,
solid line), while the field near the edges displayed a
more complex pattern (Fig. 2a, dotted line) that may
result from stronger interactions with the Petri dish
material. The spectral distribution of the electric field
was similar to that measured in the antenna, both in
center and edges of the Petri dish (Fig. 2b). The time-
independent electromagnetic field amplitude in the
<figure><img src="image_8.png" /><figcaption>Fig. 7. Effects of nanosecond electromagnetic pulses (nsEMP) on gene expression involved in cell energy sensing. Relative gene expression of (a) *SnRK1* (Sucrose nonfermenting-related kinase 1) and (b) the *TOR* (target of rapamycin) genes. Data are means of three biological independent replicates ± SE (each comprising two technical repeats) of relative expression compared to T0 after normalization using expression of reference genes (*Actin* and *GAPDH*). The dot indicates a difference (0.05 < *P* < 0.10) between unexposed (control) and ns-EMP exposed plants after a nonparametric Wilcoxon rank sum test.</figcaption></figure>

DISCUSSION

The objective of this study was to determine if
high-amplitude ultrashort electromagnetic field
pulses emitted from an antenna could induce changes
in the expression of key genes involved in the early
stages of perception of environmental stress. We set-
up an experimental system that allowed us to expose
the plants in the open volume of an anechoic
chamber, which is the condition ensuring the
minimum plant handling to avoid possible interaction
with the plant responses to nsEMP exposure. It is,
also, much closer to the situations that are encoun-
tered in civilian or military applications, where
nsEMP are emitted in the environment. The nsEMP
emitted from an antenna are, indeed, much less
commonly used than their applications through
immersed electrodes in direct contact with the
culture medium in a small, dedicated cuvette. This
last kind of equipment has the advantage of allowing
the generation of very high amplitude electromag-
netic pulses (up to several hundreds of V m⁻¹) with
rise times of a few ns to several ms [Schunck
et al., 2014], but restricts the application to cell
cultures or small biological samples. Using this kind
of equipment led to achieve various biological
responses in animal and human cells, including
membrane permeabilization [Joshi and Schoen-
bach, 2010], lower mitochondrial membrane poten-
tial [Beebe et al., 2012], cytosolic calcium increase
[Vernier et al., 2003; Semenov et al., 2013, 2015],

ROS production [Nuccitelli et al., 2013] and altera-
tion in gene expression [Zhang et al., 2014; Roth
et al, 2016].

Here we used a specialized (Koshelev) antenna
[Godard et al., 2010] that was able to deliver the high
amplitude complex nanosecond pulses. The plants
were placed along the central axis of the antenna, a
configuration shown to maximize the energy absorp-
tion [Ji et al., 2006]. Using this set-up (Fig. 1a), we
were able to expose plants to high amplitude
(237 kV m⁻¹) short duration (500 ps) EMPs that
display a very short raising time (about 280 ps),
values within the orders of magnitude for this type of
electromagnetic pulses [Schunck et al., 2014]. The
frequency distribution of the nsEMP used in the
present work (Fig. 1b) showed that it mainly accounts
for frequencies much lower than 2 GHz.

Simulation work performed with CST-Studio
software using dielectric permittivity and electrical
conductivity determined with a Diline equipment
showed that the resulting field inside the Petri dish
had a temporal signature similar to that measured in
the antenna in the center of the Petri dish (Fig. 2a,
solid line), while the field near the edges displayed a
more complex pattern (Fig. 2a, dotted line) that may
result from stronger interactions with the Petri dish
material. The spectral distribution of the electric field
was similar to that measured in the antenna, both in
center and edges of the Petri dish (Fig. 2b). The time-
independent electromagnetic field amplitude in the
Petri dish remained very low, within the range of 10⁻⁵ to 10⁻⁶ V m⁻¹, and was dependent upon the frequency (Fig. 3a–d). The resulting SAR values were therefore extremely low (Fig. 3e–h), ranging from 10⁻¹³ to 10⁻¹⁵ W kg⁻¹. These results agree with those of Cavagnaro et al. (2013) suggesting that the signal attenuation of ultrawideband pulses in human tissues was significantly induced with frequency, being of about 100 dB near 3 GHz. While being of much smaller amplitude and using emitted EMPs rather than electric field generated by immersed electrodes, the set-up used in this study (30,000 EMP of 500 ps, 237 kV m⁻¹) was previously shown to induce cellular changes in cultured MMLV murine cells with a reduction of adenosine triphosphate (ATP) content [Macaire et al., 2015]. The observation of Beebe et al. [2012] showing that short EMP (15 ns) affect mitochondria suggested that this could be due to mitochondrial dysfunction.

While many reports highlight an increase in calcium signaling after exposing living tissues to nsEMP [Vernier et al., 2003; Semenov et al., 2015], the expression level of the gene encoding the calcium-binding protein calmodulin, i.e. closely related to cytosolic calcium concentration, was statistically unchanged (*P* < 0.05) in our study (Fig. 4a), strongly suggesting that no change in calcium concentration occurred in exposed plant cells. These observations are quite surprising since several studies reported that high frequency (900 MHz), low amplitude (5 V m⁻¹) electromagnetic field caused a rapid increase in calmodulin gene expression 15 min after the end of the exposure in the apical leaf of tomato [Roux et al., 2006; Beaubois et al., 2007; Roux et al., 2008]. Longer exposure over a long period of time (6 h/day for 32 days) also caused a significant increase in calmodulin gene expression in rice leaf [Kundu et al., 2021]. The lack of change in calmodulin gene expression over the whole 180 min kinetic may indicate that the 30,000 high amplitude nanosecond pulses did not constitute a signal strong enough to trigger a molecular response in Arabidopsis, despite the very high amplitude of the electric field. The resulting low amplitude of the electromagnetic field and SAR level evoked in the Petri dish, mainly due to the ultrashort duration of the electromagnetic pulses may, at least partially, explain the absence of biological responses. A similar lack of response was observed about the expression level of *ZAT12* (Fig. 4b), a zinc-finger transcription factor widely involved in plant responses to a wide variety of environmental stimuli [Davletova et al., 2005]. Here again, low-amplitude electromagnetic fields were reported to effectively increase the gene expression

of another zinc-finger protein (LebZIP) in tomato plants [Beaubois et al., 2007; Roux et al., 2008]. It is worth noting that *ZAT12* mRNA abundance was higher in both control and exposed plants 15 min after the exposure, highlighting the necessity to carry out an unexposed control for each point of the kinetic.

Several reports pointed out the activation of ROS production and scavenging activity after exposing plants to various kinds of EMF [Tkalec et al., 2007, Sharma et al., 2009; Singh et al., 2012; Chandel et al. 2017; Stefi et al., 2018]. Here we monitored the gene expression pattern of important providers of H₂O₂ in plants, the NADPH oxidases/respiratory burst oxidase homologs [Sagi and Fluhr, 2006]. Both isoforms D and F, mainly involved in plant responses to environmental stimuli, were not affected by the exposure to nsEMP (Fig. 5a). This result was consistent with the fact that the expression of the gene encoding catalase, an enzyme involved in the bulk degradation of H₂O₂, also remained unchanged after exposing plants to nsEMP (Fig. 5b).

The expression levels of genes encoding for key enzymes of the Ascorbate Peroxidase/Glutathione cycle (APX6, GSH1, and GSH2), that are involved in the fine-tuning of H₂O₂ homeostasis, were also not affected by nsEMP exposure over the earlier points of the kinetics (Fig. 6b–d, 15–60 min). A significant difference in gene expression of *APX-1* and *APX-6* (*P* < 0.05) was however seen after 180 min of plant exposure to nsEMP (2.5- and 1.2-fold, respectively, Fig. 6a and b, 180 min), suggesting that the ascorbic acid-glutathione cycle (and thus ROS homeostasis) was affected by exposure to nsEMP. The energy state of the cell appeared to be widely affected after both high frequency (900 MHz) continuous wave in plants [Roux et al., 2008] or ns-EMP in animal cells [Macaire et al., 2015] where ATP level was rapidly (30 min) and consistently decreased after exposure, potentially triggering AMPK signaling pathway [Morotomi-Yano et al., 2012]. Here we monitored the expression level of the plant energy sensor SnRK1, known to rapidly increase in case of energetic crisis (e.g. decrease in ATP concentration) to cut-down energy consuming processes [Cho et al., 2012]. The steady-state level of SnRK1 gene expression remained low and constant all along the kinetics (Fig. 7a), showing that ATP metabolism was not affected by plant exposure to nsEMF. This result was further confirmed through the expression level of the TOR kinase that functions antagonistically from SnRK1, stimulating plant development in optimal cell-energy conditions [Xiong and Sheen, 2014; McCready et al., 2020]. The TOR kinase gene expression was not significantly changed after nsEMF
Petri dish remained very low, within the range of 10⁻⁵ to 10⁻⁶ V m⁻¹, and was dependent upon the frequency (Fig. 3a–d). The resulting SAR values were therefore extremely low (Fig. 3e–h), ranging from 10⁻¹³ to 10⁻¹⁵ W kg⁻¹. These results agree with those of Cavagnaro et al. (2013) suggesting that the signal attenuation of ultrawideband pulses in human tissues was significantly induced with frequency, being of about 100 dB near 3 GHz. While being of much smaller amplitude and using emitted EMPs rather than electric field generated by immersed electrodes, the set-up used in this study (30,000 EMP of 500 ps, 237 kV m⁻¹) was previously shown to induce cellular changes in cultured MMLV murine cells with a reduction of adenosine triphosphate (ATP) content [Macaire et al., 2015]. The observation of Beebe et al. [2012] showing that short EMP (15 ns) affect mitochondria suggested that this could be due to mitochondrial dysfunction.

While many reports highlight an increase in calcium signaling after exposing living tissues to nsEMP [Vernier et al., 2003; Semenov et al., 2015], the expression level of the gene encoding the calcium-binding protein calmodulin, i.e. closely related to cytosolic calcium concentration, was statistically unchanged (*P* < 0.05) in our study (Fig. 4a), strongly suggesting that no change in calcium concentration occurred in exposed plant cells. These observations are quite surprising since several studies reported that high frequency (900 MHz), low amplitude (5 V m⁻¹) electromagnetic field caused a rapid increase in calmodulin gene expression 15 min after the end of the exposure in the apical leaf of tomato [Roux et al., 2006; Beaubois et al., 2007; Roux et al., 2008]. Longer exposure over a long period of time (6 h/day for 32 days) also caused a significant increase in calmodulin gene expression in rice leaf [Kundu et al., 2021]. The lack of change in calmodulin gene expression over the whole 180 min kinetic may indicate that the 30,000 high amplitude nanosecond pulses did not constitute a signal strong enough to trigger a molecular response in Arabidopsis, despite the very high amplitude of the electric field. The resulting low amplitude of the electromagnetic field and SAR level evoked in the Petri dish, mainly due to the ultrashort duration of the electromagnetic pulses may, at least partially, explain the absence of biological responses. A similar lack of response was observed about the expression level of *ZAT12* (Fig. 4b), a zinc-finger transcription factor widely involved in plant responses to a wide variety of environmental stimuli [Davletova et al., 2005]. Here again, low-amplitude electromagnetic fields were reported to effectively increase the gene expression

of another zinc-finger protein (LebZIP) in tomato plants [Beaubois et al., 2007; Roux et al., 2008]. It is worth noting that *ZAT12* mRNA abundance was higher in both control and exposed plants 15 min after the exposure, highlighting the necessity to carry out an unexposed control for each point of the kinetic.

Several reports pointed out the activation of ROS production and scavenging activity after exposing plants to various kinds of EMF [Tkalec et al., 2007, Sharma et al., 2009; Singh et al., 2012; Chandel et al. 2017; Stefi et al., 2018]. Here we monitored the gene expression pattern of important providers of H₂O₂ in plants, the NADPH oxidases/respiratory burst oxidase homologs [Sagi and Fluhr, 2006]. Both isoforms D and F, mainly involved in plant responses to environmental stimuli, were not affected by the exposure to nsEMP (Fig. 5a). This result was consistent with the fact that the expression of the gene encoding catalase, an enzyme involved in the bulk degradation of H₂O₂, also remained unchanged after exposing plants to nsEMP (Fig. 5b).

The expression levels of genes encoding for key enzymes of the Ascorbate Peroxidase/Glutathione cycle (APX6, GSH1, and GSH2), that are involved in the fine-tuning of H₂O₂ homeostasis, were also not affected by nsEMP exposure over the earlier points of the kinetics (Fig. 6b–d, 15–60 min). A significant difference in gene expression of *APX-1* and *APX-6* (*P* < 0.05) was however seen after 180 min of plant exposure to nsEMP (2.5- and 1.2-fold, respectively, Fig. 6a and b, 180 min), suggesting that the ascorbic acid-glutathione cycle (and thus ROS homeostasis) was affected by exposure to nsEMP. The energy state of the cell appeared to be widely affected after both high frequency (900 MHz) continuous wave in plants [Roux et al., 2008] or ns-EMP in animal cells [Macaire et al., 2015] where ATP level was rapidly (30 min) and consistently decreased after exposure, potentially triggering AMPK signaling pathway [Morotomi-Yano et al., 2012]. Here we monitored the expression level of the plant energy sensor SnRK1, known to rapidly increase in case of energetic crisis (e.g. decrease in ATP concentration) to cut-down energy consuming processes [Cho et al., 2012]. The steady-state level of SnRK1 gene expression remained low and constant all along the kinetics (Fig. 7a), showing that ATP metabolism was not affected by plant exposure to nsEMF. This result was further confirmed through the expression level of the TOR kinase that functions antagonistically from SnRK1, stimulating plant development in optimal cell-energy conditions [Xiong and Sheen, 2014; McCready et al., 2020]. The TOR kinase gene expression was not significantly changed after nsEMF
**14 Porcher et al.**

$(P < 0.05$, Fig. 7b), being even slightly stimulated (although not statistically significant) at 60 min. These results taken together demonstrated that the plant energy status was not affected when plants were subjected to nsEMP emitted from an antenna, in contrast to what was observed after the exposure of plants to low amplitude continuous wave EMF of identical duration (10 min, 900 MHz, $5 V m^{-1}$) [Roux et al., 2008]. This result may be related to the extremely short practical duration of the pulses compared to CW exposure of similar length.

In conclusion, it appears from our results that plant exposure to 30,000 nsEMP only caused limited increase in the expression level of *APX-1* and *APX-6*, while other key genes (*TCH-1*, *ZAT12*, *CAT2*, *GSH-1*, *GSH-2*) that are otherwise rapidly activated after various kinds of environmental cues remained unchanged. It could not be excluded that different metabolic pathways may be affected after nsEMP exposure. A global gene expression such as RNA sequencing approach may allow their identification and give new insights for further investigations of the consequences of nsEMF. The influence of the nsEMP parameters (amplitude, repetition rate, duration, raising time, etc.) emitted by the Koshelev antenna on their aggressiveness toward biological samples also requires further experiments, as it could not be excluded that a threshold level could be crossed for triggering biological effects.

**ACKNOWLEDGMENTS**

The authors wish to thank the Pays-de-la-Loire Region (France) for supporting this research through the MITOWAVE program awarded to V. Procaccio, and the LRC BIOEM ("Laboratoire de Recherche Conventionnée" - LRC No. 002-2011) for granting access to the exposure core facility.

**REFERENCES**

Bai F, Gusbeth C, Frey W, Nick P. 2017. Nanosecond pulsed electric fields trigger cell differentiation in *Chlamydomonas reinhardtii*. *Biochim Biophys Acta*1859:651-661.

Bai F, Gusbeth C, Frey W, Nick P. 2020. Nanosecond pulsed electric fields modulate the expression of the astaxanthin biosynthesis genes *psy*, *crtR-b* and *bkt 1* in *Haematococcus pluvialis*. *Sci Rep* 10:15508.

Beaubois E, Girard S, Lallechere S, Davies E, Paladian F, Bonnet P, Ledoigt G, Vian A. 2007. Intercellular communication in plants: Evidence for two rapidly transmitted systemic signals generated in response to electromagnetic field stimulation in tomato. *Plant, Cell Environ* 30(7):834-844.

Beebe SJ, Fox PM, Rec LJ, Willis LK, Schoenbach KH. 2003. Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells. *FASEB J* 17(11):1-23.

Beebe SJ, Chen YJ, Sain NM, Schoenbach KH, Xiao S. 2012. Transient features in nanosecond pulsed electric fields differentially modulate mitochondria and viability. *PLoS ONE* 7(12):e51349.

Berghöfer T, Eing C, Flickinger B, Hohenberger P, Wegner LH, Frey W, Nick P. 2009. Nanosecond electric pulses trigger actin responses in plant cells. *Biochem Biophys Res Commun* 387(3):590-595.

Braam J, Davis RW. 1990. Rain-, wind-, and touch-induced expression of calmodulin and calmodulin-related genes in *Arabidopsis*. *Cell* 60(3):357-364.

Buchmann L, Frey W, Gusbeth C, Ravaynia PS, Mathys A. 2019. Effect of nanosecond pulsed electric field treatment on cell proliferation of microalgae. *Bioresour Technol* 271:402-408.

Carr L, Bardet SM, Burke RC, Arnaud-Cormos D, Leveque P, O'Connor RP. 2017. Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells. *Sci Rep* 7:41267.

Cavagnaro M, Pittella E, Pisa S. 2013. UWB pulse propagation into human tissues. *Phys Med Biol* 58(24):8689-8707.

Chandel S, Kaur S, Singh H, Batish D, Kohli R. 2017. Exposure to 2100 MHz electromagnetic field radiations induces reactive oxygen species generation in *Allium cepa* roots. *J Microsc Ultrastruct* 5:225-229.

Cho YH, Hong JW, Kim EC, Yoo SD. 2012. Regulatory functions of SnRK1 in stress-responsive gene expression and in plant growth and development. *Plant Physiol* 158(4):1955-1964.

Cucurachi S, Tamis WLM, Vijver MG, Peijnenburg WJGM, Bolte JFB, de Snoo GR. 2013. A review of the ecological effects of radiofrequency electromagnetic fields (RF-EMF). *Environ Int* 51:116-140.

Davletova S, Schlauch K, Coutu J, Mittler R. 2005. The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in *Arabidopsis*. *Plant Physiol* 139(2):847-856.

Eing C, Bonnet S, Pacher M, Puchta H, Frey W. 2009. Effects of nanosecond pulsed electric field exposure on *Arabidopsis thaliana*. *IEEE Trans Dielectr Electr Insul* 16(5):1322-1328.

Giri DV. 2004. High-Power Electromagnetic Radiators: Nonlethal Weapons and Other Applications. Cambridge, MA: Harvard University Press. pp. 29-74.

Godard A, Desrumaux L, Bertrand V, Andrieu J, Lalande M, Jecko Z, Brishoual M, Couderc V, Guillerey R. 2010. A transient UWB antenna array used with complex impedance surfaces. *Int J Antennas Propag.* 243145.

Görlach A, Bertram K, Hudecova S, Krizanova O. 2015. Calcium and ROS: A mutual interplay. *Redox Biol* 6:260-271.

Grémiaux A, Girard S, Guérin V, Lothier J, Baluška F, Davies E, Bonnet P, Vian A. 2016. Low-amplitude, high-frequency electromagnetic field exposure causes delayed and reduced growth in Rosa hybrida. *J Plant Physiol* 190:44-53.

Habauzit D, Le Quément C, Zhadobov M, Martin C, Aubry M, Sauleau R, Le Dréan Y. 2014. Transcriptome analysis reveals the contribution of thermal and the specific effects in cellular response to millimeter wave exposure. *PLoS ONE* 9(10):e109435.

Hamada Y, Furumoto Y, Izutani A, Taniuchi S, Miyake M, Oyadomari M, Teranishi K, Shimomura N, Oyadomari S. 2020. Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-
14 Porcher et al.

(P < 0.05, Fig. 7b), being even slightly stimulated (although not statistically significant) at 60 min. These results taken together demonstrated that the plant energy status was not affected when plants were subjected to nsEMP emitted from an antenna, in contrast to what was observed after the exposure of plants to low amplitude continuous wave EMF of identical duration (10 min, 900 MHz, 5 V m⁻¹) [Roux et al., 2008]. This result may be related to the extremely short practical duration of the pulses compared to CW exposure of similar length.

In conclusion, it appears from our results that plant exposure to 30,000 nsEMP only caused limited increase in the expression level of APX-1 and APX-6, while other key genes (TCH-1, ZAT12, CAT2, GSH-1, GSH-2) that are otherwise rapidly activated after various kinds of environmental cues remained unchanged. It could not be excluded that different metabolic pathways may be affected after nsEMP exposure. A global gene expression such as RNA sequencing approach may allow their identification and give new insights for further investigations of the consequences of nsEMF. The influence of the nsEMP parameters (amplitude, repetition rate, duration, raising time, etc.) emitted by the Koshelev antenna on their aggressiveness toward biological samples also requires further experiments, as it could not be excluded that a threshold level could be crossed for triggering biological effects.

ACKNOWLEDGMENTS

The authors wish to thank the Pays-de-la-Loire Region (France) for supporting this research through the MITOWAVE program awarded to V. Procaccio, and the LRC BIOEM ("Laboratoire de Recherche Conventionnée" - LRC No. 002-2011) for granting access to the exposure core facility.

REFERENCES

Bai F, Gusbeth C, Frey W, Nick P. 2017. Nanosecond pulsed electric fields trigger cell differentiation in *Chlamydomonas reinhardtii*. *Biochim Biophys Acta*1859:651-661.

Bai F, Gusbeth C, Frey W, Nick P. 2020. Nanosecond pulsed electric fields modulate the expression of the astaxanthin biosynthesis genes *psy*, *crtR-b* and *bkt 1* in *Haematococcus pluvialis*. *Sci Rep* 10:15508.

Beaubois E, Girard S, Lallechere S, Davies E, Paladian F, Bonnet P, Ledoigt G, Vian A. 2007. Intercellular communication in plants: Evidence for two rapidly transmitted systemic signals generated in response to electromagnetic field stimulation in tomato. Plant, Cell Environ 30(7):834-844.

Beebe SJ, Fox PM, Rec LJ, Willis LK, Schoenbach KH. 2003. Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells. FASEB J 17(11):1-23.

Beebe SJ, Chen YJ, Sain NM, Schoenbach KH, Xiao S. 2012. Transient features in nanosecond pulsed electric fields differentially modulate mitochondria and viability. PLoS ONE 7(12):e51349.

Berghöfer T, Eing C, Flickinger B, Hohenberger P, Wegner LH, Frey W, Nick P. 2009. Nanosecond electric pulses trigger actin responses in plant cells. Biochem Biophys Res Commun 387(3):590-595.

Braam J, Davis RW. 1990. Rain-, wind-, and touch-induced expression of calmodulin and calmodulin-related genes in *Arabidopsis*. *Cell* 60(3):357-364.

Buchmann L, Frey W, Gusbeth C, Ravaynia PS, Mathys A. 2019. Effect of nanosecond pulsed electric field treatment on cell proliferation of microalgae. Bioresour Technol 271:402-408.

Carr L, Bardet SM, Burke RC, Arnaud-Cormos D, Leveque P, O'Connor RP. 2017. Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells. *Sci Rep* 7:41267.

Cavagnaro M, Pittella E, Pisa S. 2013. UWB pulse propagation into human tissues. *Phys Med Biol* 58(24):8689-8707.

Chandel S, Kaur S, Singh H, Batish D, Kohli R. 2017. Exposure to 2100 MHz electromagnetic field radiations induces reactive oxygen species generation in *Allium cepa* roots. *J Microsc Ultrastruct* 5:225-229.

Cho YH, Hong JW, Kim EC, Yoo SD. 2012. Regulatory functions of SnRK1 in stress-responsive gene expression and in plant growth and development. *Plant Physiol* 158(4):1955-1964.

Cucurachi S, Tamis WLM, Vijver MG, Peijnenburg WJGM, Bolte JFB, de Snoo GR. 2013. A review of the ecological effects of radiofrequency electromagnetic fields (RF-EMF). *Environ Int* 51:116-140.

Davletova S, Schlauch K, Coutu J, Mittler R. 2005. The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in *Arabidopsis*. *Plant Physiol* 139(2):847-856.

Eing C, Bonnet S, Pacher M, Puchta H, Frey W. 2009. Effects of nanosecond pulsed electric field exposure on *Arabidopsis thaliana*. *IEEE Trans Dielectr Electr Insul* 16(5):1322-1328.

Giri DV. 2004. High-Power Electromagnetic Radiators: Nonlethal Weapons and Other Applications. Cambridge, MA: Harvard University Press. pp. 29-74.

Godard A, Desrumaux L, Bertrand V, Andrieu J, Lalande M, Jecko Z, Brishoual M, Couderc V, Guillerey R. 2010. A transient UWB antenna array used with complex impedance surfaces. *Int J Antennas Propag.* 243145.

Görlach A, Bertram K, Hudecova S, Krizanova O. 2015. Calcium and ROS: A mutual interplay. *Redox Biol* 6:260-271.

Grémiaux A, Girard S, Guérin V, Lothier J, Baluška F, Davies E, Bonnet P, Vian A. 2016. Low-amplitude, high-frequency electromagnetic field exposure causes delayed and reduced growth in Rosa hybrida. *J Plant Physiol* 190:44-53.

Habauzit D, Le Quément C, Zhadobov M, Martin C, Aubry M, Sauleau R, Le Dréan Y. 2014. Transcriptome analysis reveals the contribution of thermal and the specific effects in cellular response to millimeter wave exposure. PLoS ONE 9(10):e109435.

Hamada Y, Furumoto Y, Izutani A, Taniuchi S, Miyake M, Oyadomari M, Teranishi K, Shimomura N, Oyadomari S. 2020. Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-
mediated heme-regulated inhibitor (HRI) activation. PLoS ONE 15(3):e0229948.

Ji Z, Hagness C, Booske H, Mathur S, Meltz ML. 2006. FDTD analysis of a gigahertz TEM cell for ultra-wideband pulse exposure studies of biological specimens. IEEE Trans Biomed Eng 53(5):780-789.

Joshi RP, Schoenbach KH. 2010. Bioelectric effects of intense ultrashort pulses. Crit Rev Biomed Eng 38(3):255-304.

Kaur S, Vian A, Chandel S, Singh HP, Batish DR, Kohli RK. 2021. Sensitivity of plants to high-frequency electromagnetic radiation: cellular mechanisms and morphological changes. Rev Environ Sci Bio/Technol 20:55-74.

Kundu A, Vangaru S, Bhattacharyya S, Mallick AI, Gupta B. 2021. Electromagnetic irradiation evokes physiological and molecular alterations in rice. Bioelectromagnetics 42(2):173-185.

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2⁻²ΔΔCᵢ method. Methods 25(4):402-408.

Macaire S, Catrain A, Tortel S, Joly JC, Girard S, Bonnet P, Vian A. 2015. Radiated ultrashort high-power electromagnetic pulses induce ATP release in B16F10 murine melanoma cells. J Electromagn Anal Appl 07:66-74.

McCready K, Spencer V, Kim M. 2020. The importance of TOR kinase in plant development. Front Plant Sci 11:16.

Morotomi-Yano K, Akiyama H, Yano K. 2012. Nanosecond pulsed electric fields activate AMP-activated protein kinase: Implications for calcium-mediated activation of cellular signaling. Biochem Biophys Res Commun 428(3):371-375.

Müller KJ, Sukhorukov VL, Zimmermann U. 2001. Reversible electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of submicrosecond duration. J Membr Biol 184:161-170.

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mediated heme-regulated inhibitor (HRI) activation. PLoS ONE 15(3):e0229948.

Ji Z, Hagness C, Booske H, Mathur S, Meltz ML. 2006. FDTD analysis of a gigahertz TEM cell for ultra-wideband pulse exposure studies of biological specimens. IEEE Trans Biomed Eng 53(5):780-789.

Joshi RP, Schoenbach KH. 2010. Bioelectric effects of intense ultrashort pulses. Crit Rev Biomed Eng 38(3):255-304.

Kaur S, Vian A, Chandel S, Singh HP, Batish DR, Kohli RK. 2021. Sensitivity of plants to high-frequency electromagnetic radiation: cellular mechanisms and morphological changes. Rev Environ Sci Bio/Technol 20:55-74.

Kundu A, Vangaru S, Bhattacharyya S, Mallick AI, Gupta B. 2021. Electromagnetic irradiation evokes physiological and molecular alterations in rice. Bioelectromagnetics 42(2):173-185.

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2⁻²ΔΔCᵢ method. Methods 25(4):402-408.

Macaire S, Catrain A, Tortel S, Joly JC, Girard S, Bonnet P, Vian A. 2015. Radiated ultrashort high-power electromagnetic pulses induce ATP release in B16F10 murine melanoma cells. J Electromagn Anal Appl 07:66-74.

McCready K, Spencer V, Kim M. 2020. The importance of TOR kinase in plant development. Front Plant Sci 11:16.

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