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The effect of the non-ionizing radiation on exposed, laboratory cultivated maize (Zea mays L.) plants
Accepted Manuscript Title: The effect of the non-ionizing radiation on exposed, laboratory cultivated maize (Zea mays L.) plants Authors: Aikaterina L. Stefi, Lukas H. Margaritis, Nikolaos S. Christodoulakis PII: DOI: Reference:
S0367-2530(17)33217-6 http://dx.doi.org/doi:10.1016/j.flora.2017.05.008 FLORA 51131
To appear in: Received date: Revised date: Accepted date:
28-2-2017 9-5-2017 16-5-2017
Please cite this article as: Stefi, Aikaterina L., Margaritis, Lukas H., Christodoulakis, Nikolaos S., The effect of the non-ionizing radiation on exposed, laboratory cultivated maize (Zea mays L.) plants.Flora http://dx.doi.org/10.1016/j.flora.2017.05.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The effect of the non-ionizing radiation on exposed, laboratory cultivated maize (Zea mays L.) plants
Aikaterina L. Stefi a, Lukas H. Margaritis b and Nikolaos S.
Christodoulakis a* a
Section of Botany, Faculty of Biology, National and Kapodistrian University of Athens, Ilissia, Athens - 15701, Hellas (GR). b
Section of Cell Biology & Biophysics, Faculty of Biology, National and Kapodistrian University of Athens, Ilissia, Athens - 15701, Hellas (GR). *
For correspondence. E-mail: [email protected]
Article submission to:
Morphology, Distribution, Functional Ecology of Plants ISSN: 0367-2530
Copyright © Elsevier B.V. All rights reserved.
Graphical abstract
Highlights
Young corn plants exposed to long term radiation from a DECT base-unit.
No biomass reduction was observed for the exposed plants, after two weeks.
Photosynthetic pigment content seems unaltered.
After two weeks, mesophyll chloroplast structure seems not to be affected.
Bundle sheath chloroplasts severely affected, apprehending repression of a major advantage.
ABSTRACT: A series of experiments was carried out to investigate possible structural or biochemical effects on young Zea mays plants after a long-term exposure to non-ionizing, continuous radiation emitted from the base unit of a cordless DECT system. Exposed plants, compared to their normal counterparts, do not seem to be affected concerning their sprouting potential, biomass production for both the above ground part and the root, leaf structure, photosynthetic pigment content and their absorbance. The structural profile of the exposed plants seems almost identical to those of the control ones. Biomass production, photosynthetic pigments, leaf structure and chloroplast arrangement do not differ in exposed plants. What seems to be affected is the structure of the chloroplasts accommodated in the bundle sheath cells of the exposed leaves. They suffer a slight swelling of their thylakoids and an undulation of some of the thylakoid membranes. Scarcely a disruption of chloroplast envelope can be observed.
Keywords: Kranz anatomy, bundle sheath, leaf anatomy, root anatomy, chloroplast deformations, radiation
1. Introduction Ionizing radiation imposes life to a severe stress because energy absorption often leads to a biological injury. UV, X-rays and Gamma rays are electromagnetic (EM) radiations differing in frequency and, consequently, in energy (Kovács and Keresztes, 2002; Esnault et al., 2010). It has also been reported that the natural radiofrequency environment round the Globe has remained more or less the unaltered within the lifespan of the vivacious trees since before 1800 (Haggerty, 2010). The major components of this environment were broadband radio noise from space (galactic noise), from lightning (atmospheric noise), and a smaller Radio Frequency (RF) component from the sun. We may assume that the plants have evolved learning to use these environmental signals, along with visible light, in order to regulate their periodic functions. Thus, being sensitive to radiation they may also exhibit sensitivity to man-made RF fields (Haggerty, 2010). During the last decades, wireless telephones and, later on, mobile phones turned to be the most common form of communication. Therefore, the living organisms of the civilised world thrive within a “cloud” of non-ionizing radiations. The rapidly increasing use of the cellular technology resulted in an increase of electromagnetic radiations in the environment (Sharma and Parihar, 2014). Much concern is given to the effects of this radiation to human life and environmental health (Roux et al., 2006; Pietruszewski et al., 2007; Sheridan et al., 2010; Sharma and Parihar, 2014). Although plants constitute an outstanding model to study the effect of High Frequency nonionizing ElectroMagnetic Fields (HF-EMF) since their architecture (high surface area to volume ratio) optimises their interaction with the environment (Vian, et al., 2016), limited concern was given to plant reactions (Ledoigt, 2006; Roux et al., 2006;
Pietruszewski et al., 2007; Haggerty, 2010; Kumar et al., 2015) and only recently a few data became available, on the biomass production, leaf anatomy and tissue organization, overall, for two common species (Stefi et al., 2016, 2017). Having in mind previous reports on two widely used dicotelydons – Arabidopsis thaliana (L.) Heynh, as a major experimental model plant and Gossypium hirsutum L. (upland cotton) as a widely cultivated crop plant (Stefi et al., 2016, 2017) – we attempted a similar investigation on another widely used, extremely important for the global economy crop plant, Zea mays L. ssp mays. Z. mays was investigated for the abiotic stress caused by an electromagnetic field of 940 MHz (Zare et al., 2015). It was then reported that the malondealdehyde (MDA) content in the leaves of the treated plants was increased, being an indication of lipid peroxidation. Proline, also measured as a biomarker of abiotic stress and the catalase enzyme activity were all increased as a result of exposure to the electromagnetic field (EMF). EMF at 1200 MHz was also reported to inhibit early seedling growth in Z. mays, causing alterations in starch and sucrose metabolism (Kumar et al., 2015). Moreover chromosomal aberrations were observed in root tip cells of Z. mays induced by 900 MHz RF radiation (Răcuciu, 2009) while exposure times between one and eight hours, at 1 GHz radiation, on Z. mays seeds, have disruptive effects on plantlets developed from exposed seeds, regarding the young plant growth, photo-assimilatory pigments and nucleic acids contents (Răcuciu et al., 2015). Considering all the above as well as a) the fact that maize is a monocotyledon with different genetic makeup, seedling growth, tissue organization and development and photosynthetic potential, compared to A. thaliana (L.) and G. hirsutum (L.). b) monocotyledons are considered to be more tolerant to various environmental
distresses (Taylor, 1996), we attempted a crucial comparison of the ability of this species to handle DECT emitted, non-ionizing radiations to the ability of the so far thoroughly investigated dicotyledons.
2. Materials and methods 2.1. Plant Material and exposure setup Grains of Zea mays L. ssp mays were imbibed and incubated at 25 °C (70% humidity) in the dark. Soon after the coleoptile and the apex of the primary leaf appeared (5 to 7 mm protrusion) the plantlets were transferred and sown in 50 mm Jiffy-7 Peat Pellets (Jiffy Products International B.V. – U.S.A.) (Fig. 1). Twelve (6+6) Jiffy-7 pellets, with five plantlets each, were placed in each of the two Faraday cages (40 cm x 40 cm x 25 cm, covered with 0.8 mm mesh - 0.1 mm stainless steel wire) thoroughly checked, after their construction, for their ability to isolate any radiation emitted from within. The cages had a build-in light source (Philips CorePro LED bulb, 11.5 W=75 W, at 2700 K, 105 mA) producing 2500 lux radiation (Photosynthetically Active Radiation = 60 μmol m-2 s-1) at the surface of the Jiffies. Both cages were placed in a ventilated, adjustable temperature P-Selecta incubator (Model No. 2000238 – Barcelona, Spain) where they remained at 25 oC for two weeks (1st experiment). In the middle of one of the two cages, the base unit of a DECT telephone apparatus (General, Model 123) was appropriately positioned (Fig. 2). The DECT base was in a 24 h a day, 7 days a week, pulsed transmission mode, at 1882 MHz, as described elsewhere (Margaritis et al., 2014) while the light/dark programme of the chamber was adjusted to a 16/8 cycle (Stefi et al., 2016, 2017). The experiment
was repeated once again with identical setup and temperature (2nd experiment). Incubator temperature of was set at this value because germination and growth rate of Z. mays L. are reported to be optima at 25 oC (Cutforth et al., 19n86).
Radiation was measured in the two cages, while the DECT device was transmitting within one of them, with a NARDA SRM3000 (Germany) spectrum analyzer. Τhe corresponding electrical field intensity (average and peak), in each experimental setup, was measured for a 6-minute period according to ICNIRP (1998) guidelines as in Table 1. Supplementary, low precision measurements were made in the control cage with a broadband field meter (TES-92, 50 MHz - 3.5 GHz, Electromagnetic radiation detector – TES Electrical Electronic Corp. Taipei, Taiwan, R.O.C.) at the value of 490.1 mV/m. In the nearby cage (exposed), radiation reached the value of 27.46 V/m (27.460 mV/m, at 1882 MHz) (55 fold higher). 2.2. Microscopy At the end of each experiment, the Jiffies were removed from the cages and dispersed in water to release the plants. Then, the plants were washed, in order to remove any remnants of the culturing substrate from the roots, and placed on a filter paper (Figs 3 and 4) to dry at 60 oC, for three days. Each of the plants was weighed for the above ground part and the root system. Before drying, small pieces of leaves and roots were fixed for microscopy. A small part from the centre of the leaf blade was removed from three leaves taken in random, cut in to small pieces (1x1 mm) and fixed in phosphate buffered 3% glutaraldehyde (pH 6.8) at 0 oC for 2 hours (Sabatini et al., 1963). A few pieces were dehydrated in graded acetone series, critical point dried, coated with
gold and viewed with a JEOL JSM-6360 Scanning Electron Microscope. The rest of the tissue was post fixed in 1% osmium tetroxide in phosphate buffer, dehydrated in graded ethanol series and embedded in Durcupan ACM (Fluka, Steinheim, Switzerland). Semithin sections, for light microscopic observations, were obtained with an LKB 146 Ultrotome III, were placed on glass slides and stained with 0.5 toluidine blue O (in 1% borax solution), as a general stain. Ultrathin sections were placed on 100 mesh grids, double stained with uranyl acetate and lead citrate (Reynolds, 1963) and viewed with a Phillips EM-300 Transmission Electron Microscope. The same procedure was followed for the roots. Fixations were repeated after each one of the two experiments and the embedded tissues were sectioned and observed, for cross-checking the results. Literature for double fixation is cited in detail by Christodoulakis et al., (2009) and Christodoulakis et al., (2010). 2.3. Pigments protocol Photosynthetic pigments, were extracted from approximately 50 mg leaves, were extracted with 1 ml 80% acetone, overnight, at 4 oC. The supernatant was transferred to a 1-ml glass cuvette for measurement in UV/Vis Specol photometer (Zeiss). Absorbance was read at both 663.6 and 646.6 nm, corresponding to chlorophyll –a and chlorophyll –b respectively. Furthermore, absorbance of chlorophyll –c at 625 nm was also obtained. Quantification of pigment content was calculated by using molar extinction coefficients specifically for this method (Gechev et al., 2013). Chlorophyll –a, e663.6=76.79 and e646.6=18.58; chlorophyll –b, e663.6=9.79 and e646.6=47.04. The total chlorophyll content was calculated by using the following formula and normalised per fresh weight: Chlorophyll a+b=19,54A646.6 + 8,29A663.
For beta carotenes and xanthophylls no formula is available for quantification; only the absorbance value was utilised.
3. Results 3.1. Plant morphology and biomass Initially, the plants were compared morphologically. It is interesting that all grains germinated, in both plant groups, in both experiments.
Insert Plate 1, here → Comparing the control plants to their exposed counterparts, from the first stages of their life, we observed that there was no significant difference that could be observed with naked eye. The quantitative approach of the growth in the two cages is given in Table 2 with the values for the dry weight (biomass) of the remaining grain biomass, the above ground (stem and leaves) part and the roots, for the two groups, in each of the two experiments (Figs 3 and 4). 3.2. Pigment content The values for the absorbance of the five major chloroplast pigments from the control and exposed leaves are given in Fig. 5 for the first experiment and in Fig. 6 for the second experiment. In both experiments the main photosynthetic pigments seem to have been reduced in the leaves of the exposed plants yet, concerning the standard deviation of the mean, we observe that the graphs overlap so that the reduction cannot be considered as statistically significant (Figs 7 and 8). 3.2. Light microscopy
Light microscope observations in cross sections of control and exposed leaves revealed that no discernible differences can be observed at this level. Leaf thickness seems identical in both leaf types (Figs 9 and 10). Epidermal cells are also uniform while the mesophyll seems unaffected in the leaves of the exposed plants. A distinct characteristic of the leaves is the presence of sheaths of the parallel conductive bundles which are a main feature of the Kranz anatomy (yellow circles in Figs 11 and 12).
Insert Plate 2, here → In cross sections, the sheath cells (red arrows Figs 11 and 12) retain their magnitude and appear to host large chloroplasts in both the control and the exposed leaves. The chloroplasts, within the sheath cells, appear to be loaded with starch in both leaf types. Longitudinal sections of the sheath cells (red arrows Figs 13 and 14) clearly demonstrate the arrangement, magnitude and number of chloroplasts (black arrows Figs 13 and 14) for both leaf types. Differences cannot be traced. It seems interesting to point out that, in all pictures of cross or longitudinal sections of bundle sheaths, chloroplasts appear to be accommodated so as to line the cell walls across the bundle sheath (Figs 11, 12 and 13, 14). Microscopical observations of root cross sections (Figs 15, 16) revealed that no major differences can be pointed out between the two plant groups. Protoxylem elements are arranged in a similar way (black arrows in Figs 15, 16). Metaxylem, normally five or six vessels accommodated at the inner space of the vascular cylinder (red arrows in Figs 15, 16) above the root-hair zone, also seems similar in both root types. Cortex cells seem undisturbed in the roots of the exposed plants while minor differences concerning the
structure of the endodermis (yellow arrows in Figs 15, 16) are probably due to the developmental stage of this tissue.
Insert Plate 3, here → Cross sections of leaves (Figs 17 and 18) and roots (Figs 19 and 20) from both control and exposed plants, which experienced the same conditions in a second experiment with identical setup as in the first, were also investigated. As in the first experiment, no alterations or any visible damages could be observed. 3.3. Scanning Electron Microscopy Scanning Electron Micrographs (SEM) depicted the impact of radiation on the external morphology, i.e. the epidermal cells, stomata and the pili which appear on the edges of the elongated corn leaves. As it can clearly be demonstrated, the elongated epidermal cells with the curly anticlinal cell walls and the adjacent pili, at the leaf margin, appear identical in both leaf types (Figs 21 and 22). Moreover, epidermal cells and stomata, at the middle of the leaf lamella, do not present even the slightest difference (Figs 23 and 24).
Insert Plate 4, here → Finally, stomata, with their peculiar, bean shaped guard cells (typically paracytic, with two lateral subsidiary cells placed parallel to the pore), characteristic of Graminae, seem to be closed and appear totally undisturbed. The number (272±39 n/mm2 for the control and 280±44 n/mm2 for the exposed) and arrangement of these paracytic stomata seems also not to be affected while the particular surface texture of the lenticular subsidiary cells and
their surrounding epidermal cells is probably due to epicuticular waxes dissolved during tissue dehydration (Figs 25 and 26). 3.4. Transmission Electron Microscopy Transmission Electron Microscopy (TEM) revealed the structure of the chloroplasts, in detail. Two types of chloroplasts can be discerned in these leaves: those contained in the mesophyll cells (MC), and those of the bundle sheath cells (BSC) known to carry out the C4 photosynthesis. MC chloroplasts appear identical in both leaf types (Fig 27). They exhibit a lens-shaped structure, dense stroma, and typical arrangement of the well elaborated fretwork and multilayered grana. No abnormalities were observed in the MC chloroplasts of the exposed leaves. Lens-shaped BSC chloroplasts (C4) exhibit their particular structure with the extended thylakoid system and the total lack of grana (Fig 29). Our observations revealed that almost every BSC chloroplast (C4) from the exposed leaves seems to suffer an extended swelling of the thylakoids and an undulation of the thylakoid membranes (Fig 28 and 30). Scarcely a disruption of the chloroplast envelope can be observed. Mitochondria (Fig 30), microsomes and all cell membranes, in the mesophyll cells of the exposed leaves, appear unaffected.
Insert Plate 5, here → 4. Discussion Leaf cross sections, at the light microscope level, appear identical for both leaf types. Epidermal and bulliform cells do not differ. Both the mesophyll cells and the cells of the bundle sheaths appear unharmed as well. The whole structure of the Kranz tissue seems
apparently intact. In longitudinal leaf sections, bundle sheath cells appear elongated and the chloroplasts, at the extend it can be observed with optical microscope, are stained in the same way, appear intact and seem identical, in both leaf types. It seems interesting to point out that BSC chloroplasts appear to be accommodated on the cell walls across the bundle sheath, retaining a centrifugal position, as normally, in both leaf types. Chloroplast intracellular position in C4 plants is affected by environmental stresses, particularly by the extremely high light intensity (Yamada et al., 2009). This issue will be further discussed afterwards. Photosynthetic pigments, as it comes evident from the absorbance graph, are almost the same in both plants types. The exposed plants seem to present less variance than the control ones. Control plants seem to have their roots slightly more developed than those of the exposed plants while the above ground part appears more developed in the exposed plants, in both experiments (Table 2) yet, these differences, cannot be considered as significant (compare the bars Figs 5 and 6). In the 2nd experiment, although the set-up was identical, plant growth was somehow promoted thus the grain weigh was less, in both plant groups, since more of the reserves (starch, lipids and proteins) were consumed mainly from the endosperm as well as from the single cotyledon. Concerning the root, it seems that the above ground radiation does not affect the underground part of the plant. Sections of roots, at the area of primary growth and at the extension zone, from the exposed plants, do not reveal any deformations or other
differences indicating response to stress, compared to the roots of the control plants. Considering the alterations of the endodermal cells in the roots of the exposed individuals of Gossypium hirsutum (Stefi et al., 2017) as serious effects on the root structure and, probably, function, we may assume that exposed roots of Z. mays, at the early stages of the plant’s life, come out of the radiation stress without any damages. It has been reported, after an extensive investigation on the effect of the electromagnetic field in Z. mays, that certain plant’s countermeasures are launched against this type of abiotic stress (Zare et al., 2015). Among them are the increase of the malondealdehyde (MDA) content and the increased activity of catalase in the leaves. Proline content is also increased, significantly, in exposed plants (940 MHz) which is also considered to be a plant response against exposure. Moreover, recent reports indicate that electromagnetic field (EMF) at 1200 MHz inhibits early seedling growth in Z. mays, causing alterations in starch and sucrose metabolism (Kumar et al., 2015). In our experiment, under the culture and exposure conditions we selected and described in detail and the time span we exposed the young Z. mays plants, a similar effect was not observed. The chromosomal aberrations and the consequent root deformations observed in root tip cells of Z. mays, induced by 900 MHz RF radiation (Răcuciu, 2009) and the disruptive effects observed on plantlets developed from exposed seeds, concerning plant growth, photo-assimilatory pigments and nucleic acids contents, after radiation at 1 GHz (Răcuciu et al., 2015), could not even be suspected in our experiments since plantlet growth, pigment concentration and final yield seem to be the same as in the normal, control plants. A main point in this discussion has to do with the accommodation and structure of the
subcellular organelles and the chloroplasts in particular. It is reported that chloroplasts can change their intracellular positions to optimise photosynthetic activity and/or to reduce photodamage in response to light irradiation (Takagi 2003; Wada et al., 2003; Sato and Kadota, 2007) as well as modify their arrangement, in both BS cells and M cells, after exposure to various environmental stress conditions (Yamada et al., 2009). Therefore, we carefully observed the pattern of chloroplast accommodation in both cell types (BSC and MC), in both leaf types. Since no difference occurred between control and exposed leaves, we have to assume that the stress factor applied (radiation) did not affect the arrangement of the photosynthetic organelles in the exposed leaves, at least, until this stage of the plant’s life. However, our observations of chloroplast structure indicate that BSC chloroplasts in exposed leaves undergo deformations of their fretwork, although MC chloroplasts appear identical in shape and structure, in both leaf types. Investigations on various C4 plants revealed that BSC chloroplasts are severely damaged when exposed to salinity stress while most of the MC chloroplasts remain unaffected (Omoto et al., 2010). It has long ago been documented that deformations of the chloroplast membranes are the initial effects after exposure to stressing factors as i.e. air pollution (Psaras and Christodoulakis, 1987). Maize is a C4 plant and the Kranz cells of C4 plants, attain and maintain specific identities by producing the characteristic cell lineage round the conductive bundles and developing unique cell to cell communications in which certain molecules are involved. This mechanism seems to set up the extensive plant tolerance in certain, unfavourable environmental conditions (Hatch 1978; Taylor 1996). The key feature of C4
photosynthesis is the operation of a CO2 – concentrating mechanism in the leaves of C4 plants, which consists of a series of biochemical and structural modifications around the C3 photosynthetic pathway (Hatch, 1987). The most common C4 syndrome in higher plants involves the operation of two photosynthetic cycles (C 3 and C4) across two photosynthetic cell types, mesophyll (MC) and bundle sheath (BSC), which are arranged in concentric layers around the vascular bundle (Hatch, 1987). Therefore, maize mesophyll cells and bundle sheath cells differ not only morphologically but also biochemically, in concern to their photosynthetic pathways. The differences of the BSCs of C4 plants, in the pathway of carbon fixation, are considered to be an adaptation, particularly advantageous to plants growing in conditions of high light intensity and high temperature, because they prevent energetically wasteful photorespiration (Hatch, 1978). Considering all the above about the tolerance of the C4 plants and the results of our investigation, we may formulate the question: “Is this tolerance extended to confront some other environmental stresses as well???” Concluding, we could say that non-ionizing radiation emitted from devices of everyday use such as mobile phones, DECT phones, tablets, Wi-Fi routers etc, can by no means be considered as “innocent”. Our current results, recent papers for the effects on Arabidopsis thaliana and Gossypium hirsutum (Stefi et al., 2016, 2017), numerous reports from epidemiological researches correlating exposure and clinical disorders such as sleep disorders on children that use mobile phone before sleep (Van den Bulck 2007), promotion of lymphomas and leukemias in adults and children (Hardell et al., 2014) are serious reasons for further consideration.
Moreover, the effects of non-ionizing electromagnetic radiation on behavior (Divan et al., 2012), cardiovascular system (Celik and Hascalik 2004), reproduction and development (Margaritis et al., 2014), oxidative stress induction (Esmekaya et al., 2011; Manta et al., 2014), memory deficits (Fragopoulou et al., 2010, Ntzouni et al., 2011) and cancer provocation (Hardell and Carlberg 2009), strongly support the aspect that the problem is far more than serious and public anxiety seems justified.
5. Conclusions Taking in to account that: 1) The function of the C4 chloroplasts is uniquely associated with the function of stomata (Ghannoum, 2008). 2) Stomata of Z. mays are of the dumbbell – shape type. This type of stomata appears only in Graminae and is unique in structure and function. 3) The total yield in our experiments was almost similar for both control and exposed plants. 4) The photosynthetic pigment content, as measured with the UV/Vis Specol photometer, was more or less similar in both control and exposed plants. 5) Taking into consideration that maize plants are fully mature and pistillate female flowers appear after about two months while corns are harvested three months after sprouting, we may conclude that the differences between control and exposed plants are negligible in spite of the significant structural deformations of the agranal BSC
chloroplasts. Moreover, stomatal function seems not to be affected and photosynthesis (even the C4) not to be disturbed by radiation, until this stage of the plant’s life. Finally, we may point out that the deformations observed in the chloroplasts may affect the mature plant by suspending the great advantage of the C4 photosynthesis.
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LEGENDS to FIGURES Figures 1 - 8: Culture setup. 1) The two groups of pots, with the maize seedlings, before the start of the experiment. 2) The pots accommodated in the Faraday cages. The DECT base unit is in the right cage. 3) The yield after the end of the 1st experiment. 4) The yield after the 2 nd experiment. 5) Root, grain and above ground biomass after the first experiment 6) Root, grain and above ground biomass after the second experiment. 7) Absorbance of the photosynthetic pigments after the 1 st experiment. 8) Absorbance of the photosynthetic pigments after the 2 nd experiment.
Figures 9 - 16: 1st experiment. Light micrographs of fixed and sectioned tissue 9) Cross section of a normal leaf. Conductive bundles are transversely sectioned. 10) Cross section of an exposed leaf. Conductive bundles are transversely sectioned. 11) Detail of a cross sectioned normal leaf. 12) Detail of a cross sectioned exposed leaf. Notice the arrangement of the chloroplasts in mesophyll and bundle sheath cells (red arrows) in pictures 11 and 12. 13) Longitudinal section of a normal leaf across a conductive bundle. 14) Longitudinal section of an exposed leaf across a conductive bundle. Notice the arrangement of the chloroplasts (black arrows) within the bundle sheath cells (red arrows) in pictures 13 and 14. 15) Cross section of the conductive cylinder from the root of a normal plant. 16) Cross section of the conductive cylinder from the root of an exposed plant (In figs 15 and 16: black arrows = protoxylem elements, red arrows = metaxylem elements, yellow arrows = endodermal cells)
Figures 17 - 20: 2nd experiment. Light micrographs of fixed and sectioned tissue. 17) Cross section of a normal leaf. Conductive bundles are transversely sectioned. 18) Cross section of an exposed leaf. Conductive bundles are transversely sectioned. 19) Cross section of the conductive cylinder from the root of a normal plant. 20) Cross section of the conductive cylinder from the root of an exposed plant.
Figures 21 - 26: Scanning electron micrographs. Epidermal tissue arrangement from a normal (21) and an exposed (22) leaf. Read arrows point at the pili appearing at the edges of the elongated leaf. The typically paracytic stomata from normal (23) and exposed (24) leaves. Notice the triangular subsidiary cells and their surrounding epidermal cells. A detail of the dumbbell–shaped guard cells and the subsidiary (sb c) cells from a normal (25) and an exposed (26) leaf.
Figures 27 - 30: Transmission electron micrographs. 27) The structure of a mesophyll chloroplast (MC) from a control leaf. Well-constructed grana an a quite dense stroma can be observed. Mesophyll chloroplasts from the exposed leaves have identical structure. 28) A C4 chloroplast from the bundle sheath of an exposed plant. The slight swelling of their thylakoids and the undulation of some of the membranes can be observed. 29) Two C4 chloroplasts from the bundle sheath of a control leaf. The structure and arrangement of thylakoids can be observed. 30) Another C4 chloroplast suffering the same deformations as the one in Fig 28. In this picture, a sized mitochondrion appears to lean on the upper left margin of the chloroplast, possessing no structural deformations.
Table 1: Average and peak electrical field intensity, in each experimental setup, as measured for a 6-minute period. CAGE
Average
Control Exposed
0.073 V/m 2.072 V/m
Maximum integrated 0.458 V/m 11.320 V/m
Maximum peak 0.490 V/m 27.460 V/m
Table 2: The values recorded after weighing the dry mass of the plants (in both experiments). Dry weight (±standard deviation) of Zea mays L. ssp mays after 2 weeks of growth
1st experiment at 25 oC after 2 weeks of growth
grain
above ground
root
control (12 sprouts)
62,62 ±17,48 mg
75,91 ±14,42 mg
31,46 ±10,68 mg
exposed (12 sprouts)
60,78 ±13,64 mg
77,29 ±16,91 mg
29,49 ± 9,58 mg
2nd experiment at 25 oC after 2 weeks of growth
grain
above ground
root
control (12 sprouts)
53,85 ±18,12 mg
82,19 ±17,97 mg
37,40±11,55 mg
exposed (12 sprouts)
51,44 ±15,75 mg
86,88 ±19,57 mg
36,04 ±12,04 mg
S0367-2530(17)33217-6 http://dx.doi.org/doi:10.1016/j.flora.2017.05.008 FLORA 51131
To appear in: Received date: Revised date: Accepted date:
28-2-2017 9-5-2017 16-5-2017
Please cite this article as: Stefi, Aikaterina L., Margaritis, Lukas H., Christodoulakis, Nikolaos S., The effect of the non-ionizing radiation on exposed, laboratory cultivated maize (Zea mays L.) plants.Flora http://dx.doi.org/10.1016/j.flora.2017.05.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The effect of the non-ionizing radiation on exposed, laboratory cultivated maize (Zea mays L.) plants
Aikaterina L. Stefi a, Lukas H. Margaritis b and Nikolaos S.
Christodoulakis a* a
Section of Botany, Faculty of Biology, National and Kapodistrian University of Athens, Ilissia, Athens - 15701, Hellas (GR). b
Section of Cell Biology & Biophysics, Faculty of Biology, National and Kapodistrian University of Athens, Ilissia, Athens - 15701, Hellas (GR). *
For correspondence. E-mail: [email protected]
Article submission to:
Morphology, Distribution, Functional Ecology of Plants ISSN: 0367-2530
Copyright © Elsevier B.V. All rights reserved.
Graphical abstract
Highlights
Young corn plants exposed to long term radiation from a DECT base-unit.
No biomass reduction was observed for the exposed plants, after two weeks.
Photosynthetic pigment content seems unaltered.
After two weeks, mesophyll chloroplast structure seems not to be affected.
Bundle sheath chloroplasts severely affected, apprehending repression of a major advantage.
ABSTRACT: A series of experiments was carried out to investigate possible structural or biochemical effects on young Zea mays plants after a long-term exposure to non-ionizing, continuous radiation emitted from the base unit of a cordless DECT system. Exposed plants, compared to their normal counterparts, do not seem to be affected concerning their sprouting potential, biomass production for both the above ground part and the root, leaf structure, photosynthetic pigment content and their absorbance. The structural profile of the exposed plants seems almost identical to those of the control ones. Biomass production, photosynthetic pigments, leaf structure and chloroplast arrangement do not differ in exposed plants. What seems to be affected is the structure of the chloroplasts accommodated in the bundle sheath cells of the exposed leaves. They suffer a slight swelling of their thylakoids and an undulation of some of the thylakoid membranes. Scarcely a disruption of chloroplast envelope can be observed.
Keywords: Kranz anatomy, bundle sheath, leaf anatomy, root anatomy, chloroplast deformations, radiation
1. Introduction Ionizing radiation imposes life to a severe stress because energy absorption often leads to a biological injury. UV, X-rays and Gamma rays are electromagnetic (EM) radiations differing in frequency and, consequently, in energy (Kovács and Keresztes, 2002; Esnault et al., 2010). It has also been reported that the natural radiofrequency environment round the Globe has remained more or less the unaltered within the lifespan of the vivacious trees since before 1800 (Haggerty, 2010). The major components of this environment were broadband radio noise from space (galactic noise), from lightning (atmospheric noise), and a smaller Radio Frequency (RF) component from the sun. We may assume that the plants have evolved learning to use these environmental signals, along with visible light, in order to regulate their periodic functions. Thus, being sensitive to radiation they may also exhibit sensitivity to man-made RF fields (Haggerty, 2010). During the last decades, wireless telephones and, later on, mobile phones turned to be the most common form of communication. Therefore, the living organisms of the civilised world thrive within a “cloud” of non-ionizing radiations. The rapidly increasing use of the cellular technology resulted in an increase of electromagnetic radiations in the environment (Sharma and Parihar, 2014). Much concern is given to the effects of this radiation to human life and environmental health (Roux et al., 2006; Pietruszewski et al., 2007; Sheridan et al., 2010; Sharma and Parihar, 2014). Although plants constitute an outstanding model to study the effect of High Frequency nonionizing ElectroMagnetic Fields (HF-EMF) since their architecture (high surface area to volume ratio) optimises their interaction with the environment (Vian, et al., 2016), limited concern was given to plant reactions (Ledoigt, 2006; Roux et al., 2006;
Pietruszewski et al., 2007; Haggerty, 2010; Kumar et al., 2015) and only recently a few data became available, on the biomass production, leaf anatomy and tissue organization, overall, for two common species (Stefi et al., 2016, 2017). Having in mind previous reports on two widely used dicotelydons – Arabidopsis thaliana (L.) Heynh, as a major experimental model plant and Gossypium hirsutum L. (upland cotton) as a widely cultivated crop plant (Stefi et al., 2016, 2017) – we attempted a similar investigation on another widely used, extremely important for the global economy crop plant, Zea mays L. ssp mays. Z. mays was investigated for the abiotic stress caused by an electromagnetic field of 940 MHz (Zare et al., 2015). It was then reported that the malondealdehyde (MDA) content in the leaves of the treated plants was increased, being an indication of lipid peroxidation. Proline, also measured as a biomarker of abiotic stress and the catalase enzyme activity were all increased as a result of exposure to the electromagnetic field (EMF). EMF at 1200 MHz was also reported to inhibit early seedling growth in Z. mays, causing alterations in starch and sucrose metabolism (Kumar et al., 2015). Moreover chromosomal aberrations were observed in root tip cells of Z. mays induced by 900 MHz RF radiation (Răcuciu, 2009) while exposure times between one and eight hours, at 1 GHz radiation, on Z. mays seeds, have disruptive effects on plantlets developed from exposed seeds, regarding the young plant growth, photo-assimilatory pigments and nucleic acids contents (Răcuciu et al., 2015). Considering all the above as well as a) the fact that maize is a monocotyledon with different genetic makeup, seedling growth, tissue organization and development and photosynthetic potential, compared to A. thaliana (L.) and G. hirsutum (L.). b) monocotyledons are considered to be more tolerant to various environmental
distresses (Taylor, 1996), we attempted a crucial comparison of the ability of this species to handle DECT emitted, non-ionizing radiations to the ability of the so far thoroughly investigated dicotyledons.
2. Materials and methods 2.1. Plant Material and exposure setup Grains of Zea mays L. ssp mays were imbibed and incubated at 25 °C (70% humidity) in the dark. Soon after the coleoptile and the apex of the primary leaf appeared (5 to 7 mm protrusion) the plantlets were transferred and sown in 50 mm Jiffy-7 Peat Pellets (Jiffy Products International B.V. – U.S.A.) (Fig. 1). Twelve (6+6) Jiffy-7 pellets, with five plantlets each, were placed in each of the two Faraday cages (40 cm x 40 cm x 25 cm, covered with 0.8 mm mesh - 0.1 mm stainless steel wire) thoroughly checked, after their construction, for their ability to isolate any radiation emitted from within. The cages had a build-in light source (Philips CorePro LED bulb, 11.5 W=75 W, at 2700 K, 105 mA) producing 2500 lux radiation (Photosynthetically Active Radiation = 60 μmol m-2 s-1) at the surface of the Jiffies. Both cages were placed in a ventilated, adjustable temperature P-Selecta incubator (Model No. 2000238 – Barcelona, Spain) where they remained at 25 oC for two weeks (1st experiment). In the middle of one of the two cages, the base unit of a DECT telephone apparatus (General, Model 123) was appropriately positioned (Fig. 2). The DECT base was in a 24 h a day, 7 days a week, pulsed transmission mode, at 1882 MHz, as described elsewhere (Margaritis et al., 2014) while the light/dark programme of the chamber was adjusted to a 16/8 cycle (Stefi et al., 2016, 2017). The experiment
was repeated once again with identical setup and temperature (2nd experiment). Incubator temperature of was set at this value because germination and growth rate of Z. mays L. are reported to be optima at 25 oC (Cutforth et al., 19n86).
Radiation was measured in the two cages, while the DECT device was transmitting within one of them, with a NARDA SRM3000 (Germany) spectrum analyzer. Τhe corresponding electrical field intensity (average and peak), in each experimental setup, was measured for a 6-minute period according to ICNIRP (1998) guidelines as in Table 1. Supplementary, low precision measurements were made in the control cage with a broadband field meter (TES-92, 50 MHz - 3.5 GHz, Electromagnetic radiation detector – TES Electrical Electronic Corp. Taipei, Taiwan, R.O.C.) at the value of 490.1 mV/m. In the nearby cage (exposed), radiation reached the value of 27.46 V/m (27.460 mV/m, at 1882 MHz) (55 fold higher). 2.2. Microscopy At the end of each experiment, the Jiffies were removed from the cages and dispersed in water to release the plants. Then, the plants were washed, in order to remove any remnants of the culturing substrate from the roots, and placed on a filter paper (Figs 3 and 4) to dry at 60 oC, for three days. Each of the plants was weighed for the above ground part and the root system. Before drying, small pieces of leaves and roots were fixed for microscopy. A small part from the centre of the leaf blade was removed from three leaves taken in random, cut in to small pieces (1x1 mm) and fixed in phosphate buffered 3% glutaraldehyde (pH 6.8) at 0 oC for 2 hours (Sabatini et al., 1963). A few pieces were dehydrated in graded acetone series, critical point dried, coated with
gold and viewed with a JEOL JSM-6360 Scanning Electron Microscope. The rest of the tissue was post fixed in 1% osmium tetroxide in phosphate buffer, dehydrated in graded ethanol series and embedded in Durcupan ACM (Fluka, Steinheim, Switzerland). Semithin sections, for light microscopic observations, were obtained with an LKB 146 Ultrotome III, were placed on glass slides and stained with 0.5 toluidine blue O (in 1% borax solution), as a general stain. Ultrathin sections were placed on 100 mesh grids, double stained with uranyl acetate and lead citrate (Reynolds, 1963) and viewed with a Phillips EM-300 Transmission Electron Microscope. The same procedure was followed for the roots. Fixations were repeated after each one of the two experiments and the embedded tissues were sectioned and observed, for cross-checking the results. Literature for double fixation is cited in detail by Christodoulakis et al., (2009) and Christodoulakis et al., (2010). 2.3. Pigments protocol Photosynthetic pigments, were extracted from approximately 50 mg leaves, were extracted with 1 ml 80% acetone, overnight, at 4 oC. The supernatant was transferred to a 1-ml glass cuvette for measurement in UV/Vis Specol photometer (Zeiss). Absorbance was read at both 663.6 and 646.6 nm, corresponding to chlorophyll –a and chlorophyll –b respectively. Furthermore, absorbance of chlorophyll –c at 625 nm was also obtained. Quantification of pigment content was calculated by using molar extinction coefficients specifically for this method (Gechev et al., 2013). Chlorophyll –a, e663.6=76.79 and e646.6=18.58; chlorophyll –b, e663.6=9.79 and e646.6=47.04. The total chlorophyll content was calculated by using the following formula and normalised per fresh weight: Chlorophyll a+b=19,54A646.6 + 8,29A663.
For beta carotenes and xanthophylls no formula is available for quantification; only the absorbance value was utilised.
3. Results 3.1. Plant morphology and biomass Initially, the plants were compared morphologically. It is interesting that all grains germinated, in both plant groups, in both experiments.
Insert Plate 1, here → Comparing the control plants to their exposed counterparts, from the first stages of their life, we observed that there was no significant difference that could be observed with naked eye. The quantitative approach of the growth in the two cages is given in Table 2 with the values for the dry weight (biomass) of the remaining grain biomass, the above ground (stem and leaves) part and the roots, for the two groups, in each of the two experiments (Figs 3 and 4). 3.2. Pigment content The values for the absorbance of the five major chloroplast pigments from the control and exposed leaves are given in Fig. 5 for the first experiment and in Fig. 6 for the second experiment. In both experiments the main photosynthetic pigments seem to have been reduced in the leaves of the exposed plants yet, concerning the standard deviation of the mean, we observe that the graphs overlap so that the reduction cannot be considered as statistically significant (Figs 7 and 8). 3.2. Light microscopy
Light microscope observations in cross sections of control and exposed leaves revealed that no discernible differences can be observed at this level. Leaf thickness seems identical in both leaf types (Figs 9 and 10). Epidermal cells are also uniform while the mesophyll seems unaffected in the leaves of the exposed plants. A distinct characteristic of the leaves is the presence of sheaths of the parallel conductive bundles which are a main feature of the Kranz anatomy (yellow circles in Figs 11 and 12).
Insert Plate 2, here → In cross sections, the sheath cells (red arrows Figs 11 and 12) retain their magnitude and appear to host large chloroplasts in both the control and the exposed leaves. The chloroplasts, within the sheath cells, appear to be loaded with starch in both leaf types. Longitudinal sections of the sheath cells (red arrows Figs 13 and 14) clearly demonstrate the arrangement, magnitude and number of chloroplasts (black arrows Figs 13 and 14) for both leaf types. Differences cannot be traced. It seems interesting to point out that, in all pictures of cross or longitudinal sections of bundle sheaths, chloroplasts appear to be accommodated so as to line the cell walls across the bundle sheath (Figs 11, 12 and 13, 14). Microscopical observations of root cross sections (Figs 15, 16) revealed that no major differences can be pointed out between the two plant groups. Protoxylem elements are arranged in a similar way (black arrows in Figs 15, 16). Metaxylem, normally five or six vessels accommodated at the inner space of the vascular cylinder (red arrows in Figs 15, 16) above the root-hair zone, also seems similar in both root types. Cortex cells seem undisturbed in the roots of the exposed plants while minor differences concerning the
structure of the endodermis (yellow arrows in Figs 15, 16) are probably due to the developmental stage of this tissue.
Insert Plate 3, here → Cross sections of leaves (Figs 17 and 18) and roots (Figs 19 and 20) from both control and exposed plants, which experienced the same conditions in a second experiment with identical setup as in the first, were also investigated. As in the first experiment, no alterations or any visible damages could be observed. 3.3. Scanning Electron Microscopy Scanning Electron Micrographs (SEM) depicted the impact of radiation on the external morphology, i.e. the epidermal cells, stomata and the pili which appear on the edges of the elongated corn leaves. As it can clearly be demonstrated, the elongated epidermal cells with the curly anticlinal cell walls and the adjacent pili, at the leaf margin, appear identical in both leaf types (Figs 21 and 22). Moreover, epidermal cells and stomata, at the middle of the leaf lamella, do not present even the slightest difference (Figs 23 and 24).
Insert Plate 4, here → Finally, stomata, with their peculiar, bean shaped guard cells (typically paracytic, with two lateral subsidiary cells placed parallel to the pore), characteristic of Graminae, seem to be closed and appear totally undisturbed. The number (272±39 n/mm2 for the control and 280±44 n/mm2 for the exposed) and arrangement of these paracytic stomata seems also not to be affected while the particular surface texture of the lenticular subsidiary cells and
their surrounding epidermal cells is probably due to epicuticular waxes dissolved during tissue dehydration (Figs 25 and 26). 3.4. Transmission Electron Microscopy Transmission Electron Microscopy (TEM) revealed the structure of the chloroplasts, in detail. Two types of chloroplasts can be discerned in these leaves: those contained in the mesophyll cells (MC), and those of the bundle sheath cells (BSC) known to carry out the C4 photosynthesis. MC chloroplasts appear identical in both leaf types (Fig 27). They exhibit a lens-shaped structure, dense stroma, and typical arrangement of the well elaborated fretwork and multilayered grana. No abnormalities were observed in the MC chloroplasts of the exposed leaves. Lens-shaped BSC chloroplasts (C4) exhibit their particular structure with the extended thylakoid system and the total lack of grana (Fig 29). Our observations revealed that almost every BSC chloroplast (C4) from the exposed leaves seems to suffer an extended swelling of the thylakoids and an undulation of the thylakoid membranes (Fig 28 and 30). Scarcely a disruption of the chloroplast envelope can be observed. Mitochondria (Fig 30), microsomes and all cell membranes, in the mesophyll cells of the exposed leaves, appear unaffected.
Insert Plate 5, here → 4. Discussion Leaf cross sections, at the light microscope level, appear identical for both leaf types. Epidermal and bulliform cells do not differ. Both the mesophyll cells and the cells of the bundle sheaths appear unharmed as well. The whole structure of the Kranz tissue seems
apparently intact. In longitudinal leaf sections, bundle sheath cells appear elongated and the chloroplasts, at the extend it can be observed with optical microscope, are stained in the same way, appear intact and seem identical, in both leaf types. It seems interesting to point out that BSC chloroplasts appear to be accommodated on the cell walls across the bundle sheath, retaining a centrifugal position, as normally, in both leaf types. Chloroplast intracellular position in C4 plants is affected by environmental stresses, particularly by the extremely high light intensity (Yamada et al., 2009). This issue will be further discussed afterwards. Photosynthetic pigments, as it comes evident from the absorbance graph, are almost the same in both plants types. The exposed plants seem to present less variance than the control ones. Control plants seem to have their roots slightly more developed than those of the exposed plants while the above ground part appears more developed in the exposed plants, in both experiments (Table 2) yet, these differences, cannot be considered as significant (compare the bars Figs 5 and 6). In the 2nd experiment, although the set-up was identical, plant growth was somehow promoted thus the grain weigh was less, in both plant groups, since more of the reserves (starch, lipids and proteins) were consumed mainly from the endosperm as well as from the single cotyledon. Concerning the root, it seems that the above ground radiation does not affect the underground part of the plant. Sections of roots, at the area of primary growth and at the extension zone, from the exposed plants, do not reveal any deformations or other
differences indicating response to stress, compared to the roots of the control plants. Considering the alterations of the endodermal cells in the roots of the exposed individuals of Gossypium hirsutum (Stefi et al., 2017) as serious effects on the root structure and, probably, function, we may assume that exposed roots of Z. mays, at the early stages of the plant’s life, come out of the radiation stress without any damages. It has been reported, after an extensive investigation on the effect of the electromagnetic field in Z. mays, that certain plant’s countermeasures are launched against this type of abiotic stress (Zare et al., 2015). Among them are the increase of the malondealdehyde (MDA) content and the increased activity of catalase in the leaves. Proline content is also increased, significantly, in exposed plants (940 MHz) which is also considered to be a plant response against exposure. Moreover, recent reports indicate that electromagnetic field (EMF) at 1200 MHz inhibits early seedling growth in Z. mays, causing alterations in starch and sucrose metabolism (Kumar et al., 2015). In our experiment, under the culture and exposure conditions we selected and described in detail and the time span we exposed the young Z. mays plants, a similar effect was not observed. The chromosomal aberrations and the consequent root deformations observed in root tip cells of Z. mays, induced by 900 MHz RF radiation (Răcuciu, 2009) and the disruptive effects observed on plantlets developed from exposed seeds, concerning plant growth, photo-assimilatory pigments and nucleic acids contents, after radiation at 1 GHz (Răcuciu et al., 2015), could not even be suspected in our experiments since plantlet growth, pigment concentration and final yield seem to be the same as in the normal, control plants. A main point in this discussion has to do with the accommodation and structure of the
subcellular organelles and the chloroplasts in particular. It is reported that chloroplasts can change their intracellular positions to optimise photosynthetic activity and/or to reduce photodamage in response to light irradiation (Takagi 2003; Wada et al., 2003; Sato and Kadota, 2007) as well as modify their arrangement, in both BS cells and M cells, after exposure to various environmental stress conditions (Yamada et al., 2009). Therefore, we carefully observed the pattern of chloroplast accommodation in both cell types (BSC and MC), in both leaf types. Since no difference occurred between control and exposed leaves, we have to assume that the stress factor applied (radiation) did not affect the arrangement of the photosynthetic organelles in the exposed leaves, at least, until this stage of the plant’s life. However, our observations of chloroplast structure indicate that BSC chloroplasts in exposed leaves undergo deformations of their fretwork, although MC chloroplasts appear identical in shape and structure, in both leaf types. Investigations on various C4 plants revealed that BSC chloroplasts are severely damaged when exposed to salinity stress while most of the MC chloroplasts remain unaffected (Omoto et al., 2010). It has long ago been documented that deformations of the chloroplast membranes are the initial effects after exposure to stressing factors as i.e. air pollution (Psaras and Christodoulakis, 1987). Maize is a C4 plant and the Kranz cells of C4 plants, attain and maintain specific identities by producing the characteristic cell lineage round the conductive bundles and developing unique cell to cell communications in which certain molecules are involved. This mechanism seems to set up the extensive plant tolerance in certain, unfavourable environmental conditions (Hatch 1978; Taylor 1996). The key feature of C4
photosynthesis is the operation of a CO2 – concentrating mechanism in the leaves of C4 plants, which consists of a series of biochemical and structural modifications around the C3 photosynthetic pathway (Hatch, 1987). The most common C4 syndrome in higher plants involves the operation of two photosynthetic cycles (C 3 and C4) across two photosynthetic cell types, mesophyll (MC) and bundle sheath (BSC), which are arranged in concentric layers around the vascular bundle (Hatch, 1987). Therefore, maize mesophyll cells and bundle sheath cells differ not only morphologically but also biochemically, in concern to their photosynthetic pathways. The differences of the BSCs of C4 plants, in the pathway of carbon fixation, are considered to be an adaptation, particularly advantageous to plants growing in conditions of high light intensity and high temperature, because they prevent energetically wasteful photorespiration (Hatch, 1978). Considering all the above about the tolerance of the C4 plants and the results of our investigation, we may formulate the question: “Is this tolerance extended to confront some other environmental stresses as well???” Concluding, we could say that non-ionizing radiation emitted from devices of everyday use such as mobile phones, DECT phones, tablets, Wi-Fi routers etc, can by no means be considered as “innocent”. Our current results, recent papers for the effects on Arabidopsis thaliana and Gossypium hirsutum (Stefi et al., 2016, 2017), numerous reports from epidemiological researches correlating exposure and clinical disorders such as sleep disorders on children that use mobile phone before sleep (Van den Bulck 2007), promotion of lymphomas and leukemias in adults and children (Hardell et al., 2014) are serious reasons for further consideration.
Moreover, the effects of non-ionizing electromagnetic radiation on behavior (Divan et al., 2012), cardiovascular system (Celik and Hascalik 2004), reproduction and development (Margaritis et al., 2014), oxidative stress induction (Esmekaya et al., 2011; Manta et al., 2014), memory deficits (Fragopoulou et al., 2010, Ntzouni et al., 2011) and cancer provocation (Hardell and Carlberg 2009), strongly support the aspect that the problem is far more than serious and public anxiety seems justified.
5. Conclusions Taking in to account that: 1) The function of the C4 chloroplasts is uniquely associated with the function of stomata (Ghannoum, 2008). 2) Stomata of Z. mays are of the dumbbell – shape type. This type of stomata appears only in Graminae and is unique in structure and function. 3) The total yield in our experiments was almost similar for both control and exposed plants. 4) The photosynthetic pigment content, as measured with the UV/Vis Specol photometer, was more or less similar in both control and exposed plants. 5) Taking into consideration that maize plants are fully mature and pistillate female flowers appear after about two months while corns are harvested three months after sprouting, we may conclude that the differences between control and exposed plants are negligible in spite of the significant structural deformations of the agranal BSC
chloroplasts. Moreover, stomatal function seems not to be affected and photosynthesis (even the C4) not to be disturbed by radiation, until this stage of the plant’s life. Finally, we may point out that the deformations observed in the chloroplasts may affect the mature plant by suspending the great advantage of the C4 photosynthesis.
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LEGENDS to FIGURES Figures 1 - 8: Culture setup. 1) The two groups of pots, with the maize seedlings, before the start of the experiment. 2) The pots accommodated in the Faraday cages. The DECT base unit is in the right cage. 3) The yield after the end of the 1st experiment. 4) The yield after the 2 nd experiment. 5) Root, grain and above ground biomass after the first experiment 6) Root, grain and above ground biomass after the second experiment. 7) Absorbance of the photosynthetic pigments after the 1 st experiment. 8) Absorbance of the photosynthetic pigments after the 2 nd experiment.
Figures 9 - 16: 1st experiment. Light micrographs of fixed and sectioned tissue 9) Cross section of a normal leaf. Conductive bundles are transversely sectioned. 10) Cross section of an exposed leaf. Conductive bundles are transversely sectioned. 11) Detail of a cross sectioned normal leaf. 12) Detail of a cross sectioned exposed leaf. Notice the arrangement of the chloroplasts in mesophyll and bundle sheath cells (red arrows) in pictures 11 and 12. 13) Longitudinal section of a normal leaf across a conductive bundle. 14) Longitudinal section of an exposed leaf across a conductive bundle. Notice the arrangement of the chloroplasts (black arrows) within the bundle sheath cells (red arrows) in pictures 13 and 14. 15) Cross section of the conductive cylinder from the root of a normal plant. 16) Cross section of the conductive cylinder from the root of an exposed plant (In figs 15 and 16: black arrows = protoxylem elements, red arrows = metaxylem elements, yellow arrows = endodermal cells)
Figures 17 - 20: 2nd experiment. Light micrographs of fixed and sectioned tissue. 17) Cross section of a normal leaf. Conductive bundles are transversely sectioned. 18) Cross section of an exposed leaf. Conductive bundles are transversely sectioned. 19) Cross section of the conductive cylinder from the root of a normal plant. 20) Cross section of the conductive cylinder from the root of an exposed plant.
Figures 21 - 26: Scanning electron micrographs. Epidermal tissue arrangement from a normal (21) and an exposed (22) leaf. Read arrows point at the pili appearing at the edges of the elongated leaf. The typically paracytic stomata from normal (23) and exposed (24) leaves. Notice the triangular subsidiary cells and their surrounding epidermal cells. A detail of the dumbbell–shaped guard cells and the subsidiary (sb c) cells from a normal (25) and an exposed (26) leaf.
Figures 27 - 30: Transmission electron micrographs. 27) The structure of a mesophyll chloroplast (MC) from a control leaf. Well-constructed grana an a quite dense stroma can be observed. Mesophyll chloroplasts from the exposed leaves have identical structure. 28) A C4 chloroplast from the bundle sheath of an exposed plant. The slight swelling of their thylakoids and the undulation of some of the membranes can be observed. 29) Two C4 chloroplasts from the bundle sheath of a control leaf. The structure and arrangement of thylakoids can be observed. 30) Another C4 chloroplast suffering the same deformations as the one in Fig 28. In this picture, a sized mitochondrion appears to lean on the upper left margin of the chloroplast, possessing no structural deformations.
Table 1: Average and peak electrical field intensity, in each experimental setup, as measured for a 6-minute period. CAGE
Average
Control Exposed
0.073 V/m 2.072 V/m
Maximum integrated 0.458 V/m 11.320 V/m
Maximum peak 0.490 V/m 27.460 V/m
Table 2: The values recorded after weighing the dry mass of the plants (in both experiments). Dry weight (±standard deviation) of Zea mays L. ssp mays after 2 weeks of growth
1st experiment at 25 oC after 2 weeks of growth
grain
above ground
root
control (12 sprouts)
62,62 ±17,48 mg
75,91 ±14,42 mg
31,46 ±10,68 mg
exposed (12 sprouts)
60,78 ±13,64 mg
77,29 ±16,91 mg
29,49 ± 9,58 mg
2nd experiment at 25 oC after 2 weeks of growth
grain
above ground
root
control (12 sprouts)
53,85 ±18,12 mg
82,19 ±17,97 mg
37,40±11,55 mg
exposed (12 sprouts)
51,44 ±15,75 mg
86,88 ±19,57 mg
36,04 ±12,04 mg