Response of photosynthesis and electrical reactions of wheat plants upon the action of magnetic fields in the Schumann resonance frequency band

ABSTRACT Alternating magnetic fields (MF) with Schumann resonance frequencies accompanied the development of living organisms throughout evolution, but today it remains unclear whether they can have a special biological effect in comparison with surrounding non-resonant frequencies. This work shows some stimulating effect of extremely low-frequency MFs on morphometric parameters and the activity of physiological processes in wheat (Triticum aestivum L.). It is shown that the MF effect is more pronounced for transient processes – photosynthesis reactions and changes in electrical potential caused by turning on light. For light-induced electrical reactions, the dependence of the severity of the effect on the frequency of the applied MF was demonstrated. It is shown that the most pronounced effect occurs in the 14.3 Hz field, which corresponds to the second harmonic of the Schumann resonance. The predominant sensitivity of signal-regulatory systems gives reason to assume the influence of MFs with Schumann resonance frequencies on the interaction of plants with environmental factors under conditions of a changed electromagnetic environment. Such conditions can occur, for example, with an increase in lightning activity caused by climate change, which serves as the basis for the generation of Schumann resonances, and with the development of artificial ecosystems outside the Earth’s atmosphere.


Introduction
2][3][4][5] One such band is the extremely low-frequency (ELF) Schumann resonance fields.Schumann resonances are electromagnetic oscillations of the Earth-ionosphere resonator, excited by lightning discharges.These oscillations are permanently present in the atmosphere, as they are supported by global lightning activity.0][11] In addition, a radical change in the usual electromagnetic environment, including the disappearance or change of Schumann resonances, will take place for artificial ecosystems planned to be created at space stations and bases on other planets. 12aking into account the fact that living organisms have been accompanied with the Schumann resonance frequency field throughout the entire period of evolution, it has been suggested that they developed a special sensitivity to such frequencies. 34][15] The frequency dependence in the Schumann resonance band was studied in most detail on rat cardiomyocytes. 14The results indicate that the 7.8 Hz field (the first harmonic of the Schumann resonance) causes the occurrence of intracellular calcium waves and cell contraction, which does not occur when exposed to the field with other frequencies.
][18][19] Previous studies demonstrate that the effect of ELF MF is most pronounced during biological transient processes (e.g., the transition from a dark-adapted to a light-adapted state), 16 including the development of stress responses (drought-induced responses). 17In this paper, we checked whether these effects are associated with the resonant frequency or may be explained by an increased level of ELF MF in general.

Growth and exposure of plants in the magnetic field
The experiments were carried out on wheat plants (Triticum aestivum L.) variety "Daria".Plants were grown in containers with universal soil at a temperature of 24°C and a 16-hour light/8-hour dark cycle.Lighting was provided by white and fluora fluorescent lamps.
7][18][19] The component of the induction of the Earth's magnetic field located parallel to the alternating field in the coils was 19 μT, the component perpendicular to the alternating field was 9 μT, and the vertical component was 33 μT.The experiment was carried out in parallel at several field frequencies corresponding to the second Schumann resonance and its neighborhood: 10.5, 13.3, 14.3, 15.3 Hz (Figure 1), as well as in the absence of an additional field (Control).For experiments with lightinduced electrical responses, a frequency of 18 Hz was also used.The choice of the second harmonic neighborhood is due to the large biological effect obtained in previous works. 18The field magnitude at all frequencies was 18 μT, which corresponded to the magnitudes used in our experiments previously. 17,18The plants were exposed to the field throughout the entire growing period.

Registration of morphometric parameters
The length, fresh and dry weight of shoots and roots were measured on the 24th day of plant growth.To measure dry weight, plants were dried in two heating cycles lasting 3 hours at 100°C.

Registration of chlorophyll fluorescence parameters
To record the dynamics of chlorophyll fluorescence parameters, reflecting the activity of photosynthesis, an Imaging-PAM MINI PAM fluorimeter (Heinz Walz GmbH, Germany) was used.Photosynthetic parameters were calculated using the built-in software of the device. 20Saturation flashes were delivered at a frequency of once every 30 s.The photon flux density of actinic light (480 nm) was 223 μmol m −2 s −1 .
Registration of photosynthesis activity was carried out on 14-15-day-old plants.The measurements were preceded by dark adaptation lasting 20 min.The illumination period lasting 15 min.The range of 5.5-6 min after light on was used to calculate photosynthetic rates during transient processes.The measurements were made under the same magnetic field conditions under which the plants were grown.

Registration of light-induced electric reactions
Surface potentials were measured using Ag + /AgCl macroelectrodes located on the second leaves of plants at a distance of 7-10 cm from the tip of the leaf.The measuring electrodes were in contact with the leaves through threads wetted with a standard solution (0.5 mM NaCl, 1 mM KCl, 0.5 mM CaCl 2 ).The reference electrode was in contact with the roots through wet soil.Data were recorded using a high-impedance amplifiers IPL-113 (Semico, Russia) and processed on a PC in the param2 program.
Registration of light-induced electrical reactions was carried out on 14-15-day-old plants.To record light-induced reactions, plant leaves were illuminated using a LED matrix (455 nm) with an intensity of 625 µmol m −2 s −1 .A part of leaf with measuring electrode was illuminated.The measurements were preceded by dark adaptation lasting 40 min.The illumination period lasted 40 min.The amplitude of the light-induced reaction was recorded at 40 minutes after the light was turned on.The measurements were made under the same magnetic field conditions under which the plants were grown.

Statistics
Each series of experiments consisted of 30-60 repetitions; every replicate was performed on a separate plant.Statistical analysis was performed using GraphPad Prism 6 software.The mean and standard error of mean (SE) were calculated, and the normal data distribution was confirmed for all the experiments; the significance of differences was evaluated by Student t-test.Typical and averaged records are also presented in the results.

Effect of magnetic field on morphometric parameters
To assess the integral influence of magnetic fields with different frequencies on the state of plants, their morphometric parameters, such as length, fresh and dry weight of leaves and roots, were recorded.There was practically no statistically significant effect of ELF MF on morphometric parameters.Only a slight increase in root dry weight was shown in plants grown under a field with a frequency of 14.3 Hz (second harmonic of the Schumann resonance) (Figure 2).Statistically significant differences were not  revealed when comparing the parameters of control plants with those of plants exposed to other (non-resonant) frequencies.There were also no differences among all field treatments (Figure 2).

Effect of magnetic field on chlorophyll fluorescence parameters
To assess the effect of MFs with different frequencies on the intensity of physiological processes, chlorophyll fluorescence parameters, which reflect the activity of photosynthesis, were recorded.The maximum (F v /F m ) and effective (Ф PSII ) quantum yield of photosystem II and non-photochemical quenching of chlorophyll fluorescence (NPQ) were considered as the main parameters.In response to turning on the light, wheat plants develop a lightinduced reaction, during which Ф PSII and NPQ increase nonmonotonically and reach a light-adapted level (Figure 3).ELF MF did not have a statistically significant effect on the F v /F m value.In the light-adapted state, Ф PSII and NPQ showed a weak, statistically insignificant tendency to increase under the influence of the field at all frequencies (Figure 3).No differences were found among field treatments with different frequencies.
The influence of ELF MF was revealed for the transition process -the intermediate stage of development of the light-induced photosynthetic reaction before the parameters reach a stationary level.ELF MF promotes an increase in the levels of Ф PSII and NPQ compared to the control in the first minutes after turning on the actinic light.MF with frequencies of 13.3, 14.3 and 15.3 Hz significantly increases the level of Ф PSII , MF with a frequency of 10.5 Hz causes its increase as a trend.The NPQ level was significantly increased for all treatments.No frequency dependence was found for Ф PSII and NPQ during transient processes.

Effect of magnetic field on light-induced electric reaction
To assess the influence of MFs with different frequencies on the parameters of electrical reactions, their amplitude was recorded in response to turning on the light.The experiment showed that all treatments significantly increase the amplitude of the second, long-lasting wave of hyperpolarization caused by

Discussion
Chronic environmental factors, which in particular include ELF MF, cause long-term changes in plant development, 1,4,5,18,[21][22][23] The presence of such changes can be registered by integral growth parameters and the activity of basic physiological processes.In our experiments, we saw virtually no effect of ELF MF on the morphometric parameters of wheat plants.A weak stimulating effect was observed only for root weight (Figure 2).According to the literature, growth stimulation is a characteristic response of plants to the action of low-intensity MFs of various frequencies, however, a pronounced effect, as a rule, occurs for fields of higher intensity. 1,4,5,22,23he effect on morphometric parameters is based on changes in the activity of physiological processes, primarily photosynthesis.Our work did not reveal a pronounced effect of ELF MF on the F v /F m value, as well as on the Ф PSII and NPQ values in a stationary (light-adapted) state (Figure 3).Apparently, the effect on chlorophyll fluorescence parameters, as well as on morphometric parameters, is exerted by fields of higher intensity. 4,21,23Transient processes of photosynthesis, like other transient processes in the plant organism, demonstrate greater sensitivity to ELF MF. [16][17][18] However, the magnitude of the identified differences in the level of Ф PSII and NPQ is so small that it does not allow us to study the frequency dependence (Figure 3).
The light-induced electrical reaction is the only process studied in the experiment whose parameters change significantly under the ELF MF.Stimulation, like our previous work, 16 was demonstrated for the second wave of hyperpolarization, the longest (main) stage of the light-induced electrical reaction (Figure 4).A significant increase in electrical reactions is shown at all frequencies used in the experiment.It was found that the severity of the influence of the field on the parameters of electrical reactions depends on the applied frequency.The greatest increase in the amplitude of reactions was recorded for the Schumann resonance frequency of 14.3 Hz, the smallest for frequencies of 10.5 and 18 Hz, which are at the minimum of the spectrum (Figure 1).Thus, the experiment may indicate the presence of resonance for the electrical reactions of plants.The nature of the dependence of the effect on the ELF MF frequency, which was observed in our experiments, is a controversial issue.Moreover, there is not enough data in the literature to unambiguously build a consistent scheme of the influence of any MF on physiological processes.Ca 2+ , ROS and blue light receptors cryptochromes (or other flavoprotein phosphoreceptors) are the most likely participants in plant signaling cascades simultaneously involved in the formation of a light-induced electrical reaction [24][25][26][27] and in the perception of MF 1,[28][29][30][31]32 One of the likely participants in the formation of frequency dependence seems to be Ca 2+ ions. Our revious work directly demonstrated that the amplitude of lightinduced electrical reactions depends on the Ca 2+ concentration under MF with a frequency of 14.3 Hz. 16 The significant role of Ca 2+ ions in responses to MFs with Schumann resonance and its neighborhood frequencies was also shown in a number of studies performed on animal cell cultures.3,[13][14][15]33 These works show that the ELF MF-induced effect (including calcium response) may be absent at frequencies located far from resonance.For a possible explanation of this phenomenon, a mechanism is sometimes invoked that is associated with a change in the activity of Ca 2+ -binding proteins as the frequency of the alternating field approaches the frequency of the calcium cyclotron resonance at a given constant field value.[33][34][35][36] The cyclotron resonance frequency of calcium and its subharmonics can be calculated using the formula f ¼ 1 n q 2π m B DC , where n -is the subharmonic number, q -is the ion charge, m -is the ion mass, B DC -is the induction of a constant geomagnetic field.Estimates show that at midlatitudes of the Earth, the horizontal field component, which is approximately 20 μT, can provide a subharmonic frequency of the calcium cyclotron resonance, close to the frequencies of the Schumann resonances.
To explain the resonant effects under ELF MF, alternative hypotheses are also proposed.In particular, it is proposed to consider the mechanism of radical pairs in relation to the cell's own periodic processes, such as the transport of electrons along the electron transport chains of mitochondria, the frequency of which can coincide with the frequency of the applied alternating field. 37It can also be assumed that the very structure of ion channels or components of signaling systems that can influence their permeability, due to currently unknown mechanisms, has selective sensitivity to individual MF frequencies.This mechanism may be indirectly indicated by the change in the conductivity of voltage-dependent sodium and potassium channels discovered in experiments on mouse neurons under the influence of a field with a frequency close to the second harmonic of the Schumann resonance. 38Additional research should clarify the correctness of these assumptions.

Conclusion
Thus, our experiments show some stimulating effect of MFs with Schumann resonance frequencies on morphometric parameters, photosynthetic activity and the amplitude of light-induced electrical reactions in wheat plants.However, the ELF MF effect on morphometric parameters and chlorophyll fluorescence parameters was very weak, which did not allow us to study their frequency dependence.For electrical reactions, a dependence of the severity of the effect on the frequency of the applied MF was discovered.It is shown that the most pronounced effect occurs at a frequency of 14.3 Hz, which corresponds to the second harmonic of the Schumann resonance.Taken together with the data presented in the literature with similar effects for cardiomyocytes and neurons in animals, the obtained result may indicate the universality of the effects of MFs with Schumann resonance frequencies for various biological taxa.The predominant sensitivity of signal-regulatory systems gives reason to assume the influence of MFs with Schumann resonance frequencies on the interaction of living organisms with environmental factors under conditions of a changed electromagnetic environment.Such conditions can occur, for example, with an increase in thunderstorm activity caused by climate change, which serves as the basis for the generation of Schumann resonances, and with the development of artificial ecosystems outside the Earth's atmosphere.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Frequency spectrum of ELF MF based on. 6The red lines indicate the frequencies of the first, second and third harmonics of the Schumann resonance − 7.8, 14.3 and 20.3 Hz.The blue dots indicate the frequencies at which the experiment was performed: the second harmonic of the Schumann resonance (14.3 Hz), its neighborhood (13.3 and 15.3 Hz) and the regions between resonances (10.5 and 18 Hz).The color coding of the points corresponds to that in the following figures.

Figure 2 .
Figure 2. Effect of magnetic fields with frequencies of 10.5, 13.3, 14.3 and 15.3 Hz on the morphometric parameters of wheat plants.(a) and (b) -length of leaves and roots, respectively, (c) and (d) -fresh weight of leaves and roots, respectively, (e) and (f) -dry weight of leaves and roots, respectively.C -plants grown under control conditions without an additional ELF MF. n = 60.

Figure 3 .
Figure 3.Effect of magnetic fields with frequencies of 10.5, 13.3, 14.3 and 15.3 Hz on the chlorophyll fluorescence parameters in wheat plants.(a) and (b) -averaged records of the dynamics of Ф PSII and NPQ during the transition from a dark-adapted state to a light-adapted state.The inserts show fragments of the recording with transient processes in the range of 3-9 minutes.(c) and (d) -level of Ф PSII and NPQ in a light-adapted (stationary) state.(e) and (f) -level of Ф PSII and NPQ during the transition from a dark-adapted state to a light-adapted state.Ф PSII -quantum yield of photosystem II.NPQ -non-photochemical quenching of chlorophyll fluorescence.C -plants grown under control conditions without an additional ELF MF.The color coding for graphs (a) and (b) corresponds to that for graphs (c) -f).n = 36.

e2294425- 4 light (Figure 4 )
and do not affect the remaining stages of the development of the light-induced reaction.A dependence of the severity of the MF effect on frequency was discovered.It is shown that the amplitude at the resonant frequency of 14.3 Hz significantly exceeds the amplitudes at frequencies of 10.5 and 18 Hz, located in the spectrum minima between the Schumann resonances (Figure 1).

Figure 4 .
Figure 4. Effect of magnetic fields with frequencies of 10.5, 13.3, 14.3, 15.3 and 18 Hz on the parameters of light-induced electrical reactions of wheat plants.(a) -scheme for recording light-induced electrical reactions.Illumination was produced with blue light (455 nm) using an LED matrix.Measuring electrodes (E m ) were located on the illuminated areas of the plant leaf.The reference electrode was located in the root area without light.(b) -scheme for calculating the amplitude of light-induced electrical reactions of wheat plants.(c) -averaged recordings of light-induced electrical reactions under MF with frequencies of 10.5, 14.3 and 18 Hz.The dotted line indicates standard error of mean (SE).(d) -amplitudes of light-induced electrical reactions (A ER ) under MF with frequencies of 10.5, 13.3, 14.3, 15.3 and 18 Hz.C -plants grown under control conditions without an additional ELF MF. n = 30.