MAGNETITE/TARTARIC ACID NANOSYSTEMS FOR EXPERIMENTAL STUDY OF BIOEFFECTS ON ZEA MAYS GROWTH

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1 MAGNETITE/TARTARIC ACID NANOSYSTEMS FOR EXPERIMENTAL STUDY OF BIOEFFECTS ON ZEA MAYS GROWTH M. RĂCUCIU 1, D. CREANGĂ 2 1 Lucian Blaga University of Sibiu, Environmental Sciences and Physics Department, Faculty of Sciences, Applied Ecology Research Center, 5 7 Dr. I. Ratiu Street, , Sibiu, Romania, mihaela.racuciu@ulbsibiu.ro 2 Alexandru Ioan Cuza University, Biophysics and Medical Physics Laboratory, Faculty of Physics, 11 Carol I Avenue, Iasi , Romania, mdor@uaic.ro Received September 14, 2016 Abstract. Experimental study was carried out with magnetic nanosystems supplied to Zea mays cereal species. Diluted suspensions of colloidal magnetite nanoparticles (MNPs) stabilized in water were supplied in the culture medium: volume fraction ranging between 50 and 300 µl/l, equivalent with nanoparticle concentrations between 3.7 µg/ml and 22.5 µg/ml. The impact of MNPs administration was recorded at the level of: seedling growth and green tissue contents of assimilatory pigments. Photosynthesis efficacy appeared to be stimulated by MNPs as resulted from slight but statistically significant increase of the chlorophyll a/chlorophyll b ratio. Key words: nanosized magnetite; assimilatory pigments; photosynthesis efficacy; maize biotechnology. 1. INTRODUCTION The use of nanomaterials in biotechnology has considerably sustained and encouraged the applications of material science in biology. Nanobiotechnology as a hybrid discipline can be seen as cooperation of biotechnology and nanoscience, being one of the most significant areas of nowadays research with highest and ubiquitous impact on our daily life. In the last decades, nanoparticles with size typically below 100 nm resulted in continuously increasing number of applications in technical and biomedical domains [1 2]. Consequently the environmental impact of nanotechnology products generates an increased concern regarding possible side effects that engineered nanosized particles could have on the soil, water, air, microorganisms, plants and animals. Toxic effects of nanoparticles and their potential accumulation in plants and microorganisms became a current research topic, which takes into account the impact of nanoparticles on the environment biotic components. Romanian Journal of Physics 62, 804 (2017)

2 Article no. 804 M. Răcuciu, D. Creangă 2 Despite the fact that the extensive use of MNPs and nanomaterials containing MNPs (especially for biological applications [3]) is often delayed because of practical difficulty of ensuring MNP narrow size distribution with stable and reproducible characteristics, however due to low costs to large-scale production [4], such nanosystems continuously underline enlarged array of applications in the technical and life sciences. The results of various and very accurate scientific investigations allowed hypothesizing that MNPs play important roles in living body metabolism and physiology. As for vegetal organisms Khalilov et al. [5] have detected magnetite particles in five species of plants by electron paramagnetic resonance method, concluding that vegetal organisms are capable of absorbing MNPs from the soil. In consequence the design and application of new nanodevices and nanomaterials with successful implications in plant biotechnology and agriculture [6] is increasing continuously which sustains the hope of improving crop management techniques [7]. Only few articles have reported the uptake, translocation and specific localization of MNPs in plants; for example some researchers [8 9] concluded that MNPs could penetrate living plant tissues and migrate to leaf cells. According to them and other authors [10 11], nanoparticles internalization into plant populations could be done through the leaves and/or root system. Also, Gonzalez-Melendi et al. [11] showed by microscope imaging that MNPs circulate through the vascular system and thus could act as vectors for various chemicals transportation. Among other scientific reports focused on the MNPs bioeffects on environmental organisms we mention: Manoliu et al. [12] that supplied MNPs in culture medium of fungi, Voica et al. [13]; Pintilei et al. [14]; Răcuciu et al. [15] that worked with MNPs administrated to agrotechnical plant seedlings and Lacava et al. [16] that studied MNPs effects in animal organisms. The stimulatory effect of either hydrocarbons or water suspensions of MNPs upon the mitosis rate in the root meristematic cells was discussed in [17 19] which can led to further results in stimulation of plant tissue development. Other papers describing the MNPs bioeffects on plant growth evidenced positive influence in cereals, explained on the basis of iron role in the vegetal cell metabolism [20 21]. Special attention was paid to MNPs genotoxic impact based on chromosomal aberrations screening in vegetal embryos [17 19] since aberrations may result in genetic mutations if perpetuate to the next generation; but following selection of beneficial features, such genetic changes could result in useful application in plant biotechnology. Relatively small number of studies were dedicated to MNPs influence on photosynthesis processes [13 14, 22 26] revealing the stimulatory MNPs effect on the chlorophylls content, and moreover, on the plant growth. The iron oxides from controlled administrated MNPs could represent a source of iron ions for plant growth underlying new biotechnological approach. The present study is dedicated to the effect of MNPs supply on the culture medium of Zea mays seedlings. The reported results could contribute to the

3 3 Magnetite/tartaric acid nanosystems for experimental study of bioeffects Article no. 804 scientific knowledge in the field of plantlet response at the level of the assimilatory pigments and photosynthesis efficacy following the administration of magnetite/tartaric acid nanoparticles specially designed nanosystems based on Fe 3 O 4 coated with organic acid that can be naturally found in plant leaves and fruits. 2. MATERIALS AND METHODS Diluted aqueous suspension of nanosized magnetite particles coated with tartaric acid (C 4 H 6 O 6 ) were synthesized as described in Răcuciu et al. [27] by classical co-precipitation in alkali medium at about 80 C. Technological approach in Fig. 1 is presented. Fig. 1 Schematic presentation of MNPs suspension preparation technology.

4 Article no. 804 M. Răcuciu, D. Creangă 4 We assumed that magnetite nanoparticles stabilization with tartaric acid (about 6 g) could be of particular interest in plant biotechnology, since this organic molecule is found in various plants and fruits being added to many foods as antioxidant, while magnetite (Fe 3 O 4 ) is a biocompatible compound. Considering its economic importance for agriculture and food industry, the caryopses of maize (Zea mays var. everta) were chosen as biological material. In order to diminish the putative genophond variations only healthy, non damaged caryopses were selected from single plant with vigorous biological features provided by an experimental micro population; germination occurred on porous paper support in Petri dishes (50 seeds per dish), in darkness and temperature of 24 ± 0.5 C. Before the germination, biological material preparation was carried out by immersion in 0.5% sodium hypochlorite solution (ten minutes for disinfection) and repeatedly washed with deionized water. Daily supply of every sample with 7 ml diluted MNPs aqueous suspension of certain concentration was carried out for 12 days, after the germination. Seedling growth was conducted in controlled conditions of temperature (22.0 ± 0.5 C), illumination (dark/light cycle: 14 h/10 h) and 70% humidity in laboratory climate room. Volume fraction array of MNPs suspension ( µl/l equivalent with Fe 3 O 4 /C 4 H 6 O 6 concentration ranging between 3.7 and 22.5 µg/ml Table 1) was added daily to Zea mays plantlets during their early ontogenetic development for 12 days. The control samples were let to growth in the same environmental conditions only the plants were supplied with simple deionized water. Additional control group was arranged from maize seedlings grown with 0.05 g/l tartaric acid solution (TA experimental variant) correspondingly to highest tartaric acid concentration in the coated MNPs native suspension. Table 1 MNPs concentration level in the water diluted MNPs suspension samples MNPs suspension volume fraction (µl/l) MNPs concentration (µg/ml) Transmission Electron Microscopy (TEM) TESLA device was used to get MNP images, with a resolution of 1.0 nm. Magnetization curve was recorded by Gouy method, at the constant temperature (22.0 ± 0.1 C). Magnetic field intensity was measured by a Walker Scientific MG 50D Teslameter with a Hall probe. The assimilatory pigments (chlorophyll a, chlorophyll b and total carotenoid pigments) in the green tissues were assayed by spectrophotometric methods using a JASCO V530 spectrophotometer UV-VIS device provided with quartz cells of 1cm width. By Lichtenthaler & Welburn s calculation method [28], the assay of the assimilatory pigments extracts (in 80% acetone from SILAL Trading, Chemical Reagent Company, Romania) was performed. Spectrophotometric measurements were performed at the wavelengths of: 663 nm, 646 nm and 470 nm (versus

5 5 Magnetite/tartaric acid nanosystems for experimental study of bioeffects Article no. 804 acetone 80%). Plantlet individual length was measured with 0.1 cm precision using simple ruler. Statistic analysis of the experimental data, resulted from three repetitions of the whole experiment, was accomplished by means of one way ANOVA test working with MsExcell soft package to evaluate reliability of MNP induced changes with the significance criterion of 0.05 (p value). Each repeated experiment had own control sample. Statistic analysis of plantlet length was accomplished with Student t-test and confidence levels P = 90%, 95% and 99%. 3. RESULTS AND DISCUSSIONS MNPs of quasi-spherical shape were evidenced by TEM imaging of 10 4 diluted suspensions. Average particle diameter of about 8.9 nm and particle dimensional distribution between 3 and 20 nm were found based on TEM image measurement. In Fig. 2 the distribution histogram of data provided by TEM microphotograph can be seen. Coated MNPs native suspension had nanoparticles per ml and saturation magnetization of 2.4 ka/m (data published previously in [27]). Fig. 2 Dimensional histogram of MNPs diameter (d) and TEM microphotograph colloidal MNPs. Maize seedling growth was daily checked for 12 days. According to direct visual inspection, it was presumed that brown spots observable after the ten days of seedling growth (Fig. 3) could be assigned to toxicity symptoms (necrosis) induced by MNP concentrations higher than about 11 µg/ml.

6 Article no. 804 M. Răcuciu, D. Creangă 6 Fig. 3 Left Control sample photo (untreated sample, the seedlings growth was ensured only with deionised water supply). Right Plantlets exhibiting possible toxic effects induced by MNPs supply (15 µg/l and respectively, 22.5 µg/l). According to Kampfenkel et al. [29], one could suppose that iron excess could trigger excessive oxidative stress in leaf cells leading to localized necrosis spots. In the Fig. 4 the average seedling length graph is presented. For relatively high MNPs concentration (> 7.5 µg/ml) stimulatory effect on the maize seedling growth was recorded. Fig. 4 The average length of Zea mays plantlets; Student t test analysis: P = 90% confidence level. (* statistically significant, ** not statistically significant) (TA control sample grown only with tartaric acid). Over 50% increase compared to the control was evidenced in the maize sample corresponding to about 11 µg/ml; for higher MNPs concentrations (15 µg/l and respectively, 22.5 µg/l) the stimulatory bioeffect diminished gradually reaching

7 7 Magnetite/tartaric acid nanosystems for experimental study of bioeffects Article no. 804 the level of the control sample; small changes in TA control (seedlings supplied only with tartaric acid (TA) corresponding to highest MNPs suspension concentration) and lowest MNPs concentration tested in this experiment (3.7 µg/ml) had no statistical significance. From these results it seems that during early ontogenetic stages the maize growth could be significantly influenced by MNPs supply with positive results regarding evident phenotypic feature which is seedling length. Since vegetal organism growth is based on photosynthesis complex process next analyses were focused on chlorophyll a, chlorophyll b and carotenoid pigment contents in the green tissue. According to Fig. 5 graphical plots, the chlorophyll a level, the main photosynthesis pigment, was found increased for relatively small MNP concentrations used in this experiment µg/ml (20.7 % increase for the sample supplied with 3.7 µg/ml) comparatively to the control sample (p < 0.05)). But, for relatively high MNPs suspension concentrations, the chlorophyll a level was found decreased, (with 13.2 % for the samples supplied with 15 µg/ml and respectively 22.5 µg/ml MNPs suspension). Similar response resulted for the other two analyzed pigments: chlorophyll b content was enhanced with no more than 10% for lowest MNPs concentration while for corresponding total carotene content highest average value estimated was of about 8% (p < 0.05); for MNPs concentrations over 15 µg/ml pigment levels were diminished with about 8.5 9% (standard deviation was of 5.5%). For all three types of pigments the TA control sample showed only non-significant variation. Fig. 5 Assimilatory pigment levels in Zea mays plantlets (Chl a the content of chlorophyll a, Chl b the content of chlorophyll b, Car the content of total carotenoid pigments) (* statistically significant, ** statistically non significant) (TA control sample grown only with diluted tartaric acid supply).

8 Article no. 804 M. Răcuciu, D. Creangă 8 The total assimilatory pigments content was also calculated which presented the same variation trend as that observed for chlorophyll levels. The chlorophyll ratio (chlorophyll a/chlorophyll b) in Fig. 6 is represented. This biochemical parameter can be taken as indirect indicator on the photosynthesis process efficiency [30] which provides indirect information on the functioning of enzymatic aggregates from Light Harvesting Complex II (LHC II) of the photosynthetic system II located in the chloroplasts membranes. According to Fig. 6, chlorophylls ratio increased up to 5 % when the MNP concentration was raised from 3.7 µg/ml to about 11 µg/ml (although comparable with standard deviation the maximum increase still was statistically significant: p < 0.05). This can be taken as a conclusive proof of the capacity of the MNPs to influence the LHC II enzyme system. But the attempt to shape an explanatory phenomenological description based on the above results should involve nanosized iron oxides as well as iron ions themselves. As shown by mentioned authors of [11] MNPs can circulate through the vascular system; additionally the authors of [31] underlined that plant tissues are able to receive magnetic nanoparticles by means of the plasmodesmata channels. Plasmodesmata small channels that directly connect the cytoplasm of neighbouring plant cells to each other are living bridges between cells, with important role in cellular communication. The average diameter of plasmodesmata tubes is of 50 nm with variation between 20 and 200 nm. Thus MNPs with diameter around 10 nm like most frequent ones in our experiment probably penetrate bio-membrane system; even larger particles can pass through according to Oparka [32] that reported that in the case of cell-to-cell transport of viruses, plasmodesmata can be dilated with the help of movement proteins. Fig. 6 The effects of volume fraction of magnetic fluid solution added in the plants culture medium on chlorophylls ratio (Chl a/chl b) (* statistically significant, ** not statistically significant) (TA control sample grown only with tartaric acid).

9 9 Magnetite/tartaric acid nanosystems for experimental study of bioeffects Article no. 804 But there are also other aspects that should be discussed related to this ultrastructural plant system. First, during MNPs circulation trough suitable size plasmodesmata channels they could impede usual protein normal movements and implicitly their metabolic function could be impaired. Second, according to Rabaev et al. [33] plasmodesmata geometrical plasticity could result in minor dimensional differences of small channels diameter favoured by some proteins property to change their conformation thus the smallest plasmodesmata channels could impede some internalized MNPs to pass from one cell to another. Evidently, MNPs blockage could occur in some intercellular conducts mainly some of them, with larger size, could remain stocked in the cell cellulose wall, or even in tight contact with bio-membranes. In such situations their superparamagnetic properties could influence locally the transmembrane ion flows (magnetic field influence on the electric ion currents); consequently ion signalling pathways could be disturbed with influence on cell biochemistry including assimilatory pigment synthesis or the synthesis of constitution proteins important for plant tissue growth. This is also assumed by Hughes et al. [34] who hypothesized that MNPs that remained attached to cellular membranes could influence the conductivity of mechanosensitive ion channels resulting further in cell biochemistry modulation. As for the magnitude of local magnetic field around MNPs, Binhi [35] estimated that the magnetic moment of MNPs found in living organisms is times higher than the elementary magnetic moment known in physics so that the corresponding magnetic field energy at environmental temperature is higher than the thermal movement energy taken as reference, k B T (k B Boltzmann s constant; T absolute temperature). This could lead to significant spatial restrictions of electric charged ions that normally circulate through cell membranes with well defined signalling roles; possibly assimilatory pigment synthesis or the protein accumulation are indirectly modulated by MNPs local embedding in vegetal cell ultrastructural elements. MNPs that succeeded to be internalized on vegetal cells through endocytosis could be further degraded by lysosomal digestion with release of ferric and ferrous ions [36 37]. Iron is needed for many cellular processes being present in cytochromes that catalyze redox reactions in the mitochondrial membranes or in peroxidasic enzymes like catalase that controls partially the level of toxic hydrogen peroxide. Iron depletion in the large area cultivated terrains is known to compromise biological quality of grown cereals or wine wards. Vegetal cells are therefore provided with suitable mechanism of iron storage in the form of complex combinations known as phytosiderophores; externally originating Fe +3 ion is probably changed to Fe +2 ion, significantly more hydrosoluble. In this context iron ions supply from digested MNPs could sustain plant growth in the experimentally designed arrangement where no other nutrient was additionally used. On the other side there is no doubt that iron excess could trigger cytotoxicity mechanisms due to catalytic Fenton reactions controlled by free iron ions and

10 Article no. 804 M. Răcuciu, D. Creangă 10 resulting in reactive oxygen species that overwhelm defense cellular mechanisms dedicated to balance peroxidative damages (Fig. 7). In our experiment control samples can be considered as iron deficient plantlets and thus the increased chlorophyll synthesis (Fig. 5) and fresh mass accumulation (Fig. 4) for some MNPs supplied samples was not surprising while the diminished levels of both assimilatory pigments and plant length for highest MNP concentration (over 22.5 µg/ml) could be correlated with toxic effect of iron excess. We mention that different maxima of chlorophyll graphs and plant length variation were also expected since photosynthesis is not the only process involved in plant metabolism so that accumulation of fresh biomass seems to be stimulated by higher MNPs concentration than that corresponding to chlorophyll synthesis and apparent photosynthesis efficacy. Fig. 7 MNPs digestion with iron ion release in plant cell. According to Jiang et al. [38] that measured in vivo gas exchange and chlorophyll fluorescence, iron-deficient plants exhibit considerably lower efficiency of energy capture by open PSII reaction centers so that lower photosynthesis apparent efficacy in our control plantlets should be expected. Mentioned authors advanced the hypothesis that both the electron donor and the electron acceptor sides of PSII complex were impaired by iron deficiency. According to Perriera et al. [39] that applied non-invasive measurements on photosynthesis, photorespiration and chlorophyll a fluorescence and content photosynthesis diminution at moderate Fe 2+ concentrations can be attributed to stomatal limitations while for relatively high concentrations both stomatal and nonstomatal limitations would be involved. Also the plant differentiated photosynthetic sensitivity to iron ion levels was underlined by the mentioned authors.

11 11 Magnetite/tartaric acid nanosystems for experimental study of bioeffects Article no CONCLUSIONS The highest growth stimulation in 12 days old seedlings was found for the MNPs concentration of about 11 µg/ml consisting in over 50% increasing in average plant length while for higher concentrations brown spots were evidenced suggesting toxic effect on the maize plantlets. Chlorophyll a level was found enhanced with up to 20% for MNPs concentrations beyond 11 µg/ml but for higher concentration inhibitory effect of MNPs on the assimilatory pigment biosynthesis was observed as denoted by chlorophyll a level diminution with 15 20% compared to the control samples. Similar biochemical response was obtained for chlorophyll b while for carotenoid pigments less evident changes were recorded. Photosynthesis efficacy appeared to be stimulated by MNPs as resulted from slight but statistically significant increase of the chlorophyll a/chlorophyll b ratio. MNPs uptake and lysosomal digestion was presumed to occur in the plant cells with further iron ions release that had catalytic action with influence on cytoplasm redox processes affecting chlorophyll synthesis as well. Possible biotechnological tool based on low costs and available materials could be designed to conduct the growth of cereal plantlets for enhancing some biological parameters with MNPs. REFERENCES 1. O.V. Salata, J. Nanobiotech., 2 (3), 1 6 (2004). 2. Z. Liu, F. Kiesling, J. Gatjens, J. Nanomater, 2010, 1 15 (2010). 3. W.J. Stark, Nanoparticles in Biological Systems. Angewandte Chemie International Edition, 50 (6), (2011). 4. C. Suryanarayana, C.C. Koch, Hyperfine Interact., 130 (1 4), 5 44 (2000). 5. R.I. Khalilov, A.N. Nasibova, V.A. Serezhenkov, M.A. Ramazanov, M.K. Kerimov, A.A. Garibov, et al., Biophysics., 56 (2), (2011). 6. G. Scrinis, K. Lyons, Int. J. Sociol. Food Agric., 15, (2007). 7. C. Moraru, C. Panchakapesan, Q. Huang, P. Takhistov, S. Liu, J.L. Kokini, Food Technol., 57, (2003). 8. E. Corredor, P.S. Testillano, M.J. Coronado, P. González-Melendi, R. Fernández-Pacheco, C. Marquina, et al., BMC Plant Biol., 9 (45), 1 11 (2009). 9. C.M. Rico, S. Majumdar, M. Duarte-Gardea, J.R. Peralta-Videa, J.L. Gardea-Toressdey, J. Agr. Food Chem., 59 (8), (2011). 10. H. Zhu, J. Han, J. Xiao, Y. Jin, J. Environ. Monit., 10, (2008). 11. P. González-Melendi, R. Fernández-Pacheco, M.J. Coronado, E. Corredor, P.S. Testillano, M.C. Risueño, et al., Ann. Bot., 101 (1), (2008). 12. Al. Manoliu, I. Antohe, d. E. Creanga, C. Cotae, J. Magn. Magn. Mater., 201 (1), (1999). 13. C. Voica, L. Polescu, D.A. Lazar, Rev. Roum. Biol., 48 (1 2), 9 15 (2003). 14. M. Pintilie, L. Oprica, M. Surleac, C. Dragut Ivan, D.E. Creanga, V. Artenie, Rom. J. Phys., 51 (1 2), (2006).

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