- Nano Express
- Open Access
Impact of Metal Nanoform Colloidal Solution on the Adaptive Potential of Plants
© Taran et al. 2016
- Received: 27 November 2015
- Accepted: 2 February 2016
- Published: 15 February 2016
Nanoparticles are a known cause of oxidative stress and so induce antistress action. The latter property was the purpose of our study. The effect of two concentrations (120 and 240 mg/l) of nanoform biogenic metal (Ag, Cu, Fe, Zn, Mn) colloidal solution on antioxidant enzymes, superoxide dismutase and catalase; the level of the factor of the antioxidant state; and the content of thiobarbituric acid reactive substances (TBARSs) of soybean plant in terms of field experience were studied. It was found that the oxidative processes developed a metal nanoparticle pre-sowing seed treatment variant at a concentration of 120 mg/l, as evidenced by the increase in the content of TBARS in photosynthetic tissues by 12 %. Pre-sowing treatment in a double concentration (240 mg/l) resulted in a decrease in oxidative processes (19 %), and pre-sowing treatment combined with vegetative treatment also contributed to the reduction of TBARS (10 %). Increased activity of superoxide dismutase (SOD) was observed in a variant by increasing the content of TBARS; SOD activity was at the control level in two other variants. Catalase activity decreased in all variants. The factor of antioxidant activity was highest (0.3) in a variant with nanoparticle double treatment (pre-sowing and vegetative) at a concentration of 120 mg/l. Thus, the studied nanometal colloidal solution when used in small doses, in a certain time interval, can be considered as a low-level stress factor which according to hormesis principle promoted adaptive response reaction.
- Superoxide dismutase
- Lipid peroxidation
- Factor of antioxidant activity
Abiotic stress is produced by a series of factors like extreme temperatures, chemical compounds, water deficit, and an excess of heavy metals. Metabolic processes in plants continually produce reactive oxygen species (ROS) in structures like chloroplasts, mitochondria, peroxisomes, the endoplasmic reticulum (ER), and plasma membranes . ROS is controlled by various enzymatic and non-enzymatic antioxidative systems. Enzymatic antioxidants include catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), superoxide dismutase (SOD), and enzymes that detoxify lipid peroxidation (LP) products, and non-enzymatic antioxidants include ascorbic acid (AA), glutathione (GSH), tocopherols (TOCs), carotenoids (CARs), and phenolic compounds. In addition, an array of enzymes, such as monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), is needed for the regeneration of the active forms of the antioxidants.
The unique properties of nanomaterials and nanoclusters formed are widely used in various fields of activity now [2, 3]. The metastable unbalanced state of nanoparticles does not allow the prediction of their influence upon the physiological and biochemical processes taking place in plants; therefore, the very urgent problem of identifying the degree of stress impact in a wide range of concentrations is taking place today. There is no single opinion on the impact of nanoparticles upon physiological and biochemical processes in the literature available—both positive and negative effects are noted. The pro-oxidant effect of synthesized fullerene-containing (10–6 mol/l by C60) supramolecular composites on the lipid peroxidation processes has been revealed [4, 5].
It is known that nanoparticles have positive morphological effects like the enhancement of seed germination rates and the improvement of root and shoot formation and their ratio, as well as the accumulation of vegetative biomass of seedlings in many crop plants . Nanoparticles’ influence on the cell level thus increases the pace of physiological processes in plants. The nanoparticles of zinc, cuprum, iron, etc. revealed by now are up to 40 times less toxic than the salts [7, 8]. They are gradually absorbed while their ionic forms are immediately included into the biochemical reactions. Nanoparticles take part in the electron transfer in plants and thus increase the activity of plant enzymes, intensify photosynthesis processes, and have a direct influence on the plant mineral nutrition [9–11]. The colloidal solutions containing biologically active metals are now being widely used along with traditional biological preparations. There are preliminary conclusions about the positive effects of these preparations on the productivity and plant resistance to adverse environmental factors . It was found also that various nanoparticles exhibit antioxidant enzyme-like ability: nCeO2 and nFe3O4 reveal catalase activity; nCeO2, nFe3O4, nCo3O4, nMnO2, nCuO, and nAu exhibit peroxidase activity; and nCeO2, nPt, and fullerene demonstrate superoxide dismutase property . There is an opinion that the presence of nonpolar environments seems to enhance the reactivity of the fullerene molecule toward OH radicals compared to the gas phase. Energetic considerations show that, once a first radical is attached to the fullerene cage, further additions are increasingly feasible, suggesting that fullerene can act as OH radical sponges . Unfortunately, it is next to impossible to separate these activities in experiments: nTiO2-A enhanced the activities of SOD, CAT, APX, and GPX in spinach  and GPX, SOD, and CAT in Lemna minor . It is known also about the photoreduction activity of Vigna radiata isolated chloroplasts exposed to nMn. Nanoparticles are revealed to modulate the activity of photosystem II (PSII) by enhancing the splitting of water and oxygen evolution thus improving the photophosphorylation activity of the electron transport chain. The enhanced activity of the CP43 protein of a PSII Mn4Ca complex influenced better phosphorylation in the electron transport chain in a nMn-treated chloroplast .
Thus, the role of nanoparticles’ chemical attributes on the modulation of the antioxidant defense system in plants is not clear and a topic worthy of attention. Based on this information, the purpose of our research was to study the effect of different concentrations (120 and 240 mg/l) of trace element (Ag, Cu, Fe, Zn, Mn) nanoform colloidal solution as biogenic minerals used in agricultural technologies for plant nutrition, regulation of physiological processes, and the development of plant adaptive reactions in a field experiment.
The object of the study was soybean plants of the early ripening variety (Annushka—Ukrainian selection, PC “Soeviy Vik”). The investigation was performed in field conditions. The soil of the experimental field was typical black soil with humus content in the arable soil layer equal to 4.38–4.53 %, pH of salt extract—6.9–7.3, nitrogen—0.27–0.31 %, phosphorus—0.15–0.25 %, and potassium—2.3–2.5 %. The farming equipment is common to the northern forest steppe of Ukraine. The treatment of seeds and plants with a colloidal solution of metal nanoparticles was performed under the following scheme: 1—control, treatment with water; 2—pre-sowing seed treatment with complex metal colloids at a concentration of 120 mg/l; 3—pre-sowing seed treatment with complex metal colloids at a concentration of 240 mg/l; and 4—pre-sowing seed treatment combined with vegetative treatment (spraying in the budding stage) with complex metal colloids at a concentration of 120 mg/l. Analysis of control and treated soybean plants was carried out at the stage of three true leaves and at vegetation after treatment with nanoparticle solutions.
Characteristics of the colloidal solution of metal nanoparticles 
The average diameter, nm
Cu, CuO, Cu2O
Fe, Fe2O3, Fe3O4
Mn, Mn3O4, Mn2O7
The photosynthetic tissue of soybean plants (300 mg) were washed with distilled water and homogenized with 1.5 ml of 50 mM phosphate buffer (pH 7.8). The homogenate was centrifuged for 15 min at 12 000 rpm at 4 °C. The supernatant was stored at 4 °C and used to determine enzyme activity and protein content.
The activity of SOD (EC 18.104.22.168) was determined according to the ability to inhibit reduction of nitroblue tetrazolium (NBT) at λ = 560 nm. The unit of enzyme activity was 50 % inhibition of formazan formation. SOD activity is expressed in arbitrary units per milligram protein .
The activity of CAT (EC 22.214.171.124) was determined according to Aeby . Activity was expressed as arbitrary units per milligram protein.
Determining the Level of LP
The level of lipid peroxidation was evaluated by the accumulation of thiobarbituric acid reactive substances (TBARSs) according to Kumar and Knowles  with modifications. The intensity of lipid peroxidation (LP) was evaluated by the content of malonic dialdehyde that was determined by 2-thiobarbituric acid reaction. Photosynthetic tissues (200 mg) were homogenized with a small amount (1.5 ml) of Tris-NaCl buffer (pH 7.6), and the volume of the extract was adjusted to 3 ml. To the resulting homogenate, 1 ml of 0.5 % solution thiobarbituric acid (TBA) and 2 ml of 20 % trichloroacetic acid (TCAA) were added. Test tubes with homogenates withstand 30 min in a boiling water bath, followed by centrifugation at 3000 rpm for 10 min. Light absorption was recorded at λ = 533 nm. Content of TBARS was expressed in micromoles of malondialdehyde using a molar extinction coefficient 1.55 × 105 cm−1 M−1.
The determination of dry biomass was performed according to GOST 16932-93. The determination of protein was performed based on the method of Bradford . Statistical analysis of the data obtained was performed with the help of program Statistica 6.0. The differences are considered significant at a value P ≤ 0.05.
Thus, the most “antistress” treatment judging upon TBARS was variant 3—pre-sowing seed treatment with complex metal colloids at a concentration of 120 mg/l, whereas in variant 2 the content of TBARS overcame control despite maximal SOD activity. On the other side, taking into account all indexes, the most successful combination was applied in variant 4—pre-sowing seed treatment combined with vegetative treatment (spraying in the budding stage) with complex metal colloids at a concentration of 120 mg/l. It is worthy to note that chlorophyll decrease did not take place here.
Data available in the literature do not contradict ours. Significant reduction of H2O2 accumulation and lipid peroxidation (TBARS content decreases, same as the index of ROS production) at 25 and 50 parts per million (ppm, mg/l, or mg/kg) of nAg-treated (25–400 ppm) 7-day-old Brassica juncea seedlings was observed. Improved photosynthetic quantum efficiency and higher chlorophyll content were recorded in leaves of treated seedlings, whereas levels of TBARS and H2O2 decreased herein. Nanoparticle treatment induced the activities of specific antioxidant enzymes like GPX, CAT, and APX, causing the ROS level to decrease. Besides an increase in chlorophyll a content, PSII quantum efficiency (Fv/Fm) was observed at 25, 50, and 100 ppm nAg treatment. The higher quantum efficiency (Fv/Fm) shows that the greater number of reaction centers are “open” to hold a light reaction that reduces the probability of the ROS generation. It was suggested that nAg increased the efficiency of redox reactions, based on the ability of nAg to act as an electron transfer center increasing the efficiency of redox reactions .
Both forms of chlorophyll are involved in light harvesting, whereas special forms of only chlorophyll a are linked into energy-processing centers of photosystems. In strong light, photons are abundant, consistent with a substantial capacity for energy processing by leaves (hence the higher chlorophyll a/b ratio). In weak light, optimization of leaf function calls for greater investment of leaf resources in light harvesting rather than energy processing. Because of how a photosystem looks like (chlorophyll a in the middle, chlorophyll b at the periphery), the plant can only increase the amount of chlorophyll b (there is no way to throw some molecules in the middle of the photosystem). So as it accumulates more chlorophyll b, the ratio drops. The chlorophyll a/b ratio can be a useful indicator of plant state, because this ratio should be positively correlated with the ratio of PSII cores to LHCII. It contains the majority of chlorophyll b and has therefore a lower chlorophyll a/b ratio than other chlorophyll-binding proteins of PSII . Therefore, chlorophyll a/b ratio can be applied for the rapid detection of plant stress in arid ecosystems .
Concerning stress action, it is known that any stress causes oxidative stress followed by damage of the photosynthetic apparatus. For example, heavy metals (Pb, Cu, Cd, Hg) decreased the total chlorophyll content in the leaves of bean seedlings progressively, but the chlorophyll a/b ratio increased slightly with increasing concentrations of heavy metal . The strong decrease of chlorophyll content was associated with a twofold increase of the chlorophyll a/b ratio in salt-stressed spinach leaves . Some types of stress increase the ratio of chlorophyll a to chlorophyll b; other stresses reduce it . Our data allow us to conclude that in variants 2 and 4 plant photosynthesis was affected to a lesser extent.
Carotenoids also participate in photosynthetic energy transduction as an “accessory” to primary pigments (chlorophylls) and perform several functions in photosynthetic membranes. The most important is preventing the formation of singlet oxygen and protecting chlorophylls by quenching their triplet states. Carotenoids play also a central role for chlorophyll-binding proteins of both the antenna system and the reaction center and for D1 protein assembly during its turnover while forming functional PSII complexes. Inhibitors of carotene biosynthesis led to the loss of both PSII activity and D1 protein, indicating the requirement of β-carotene synthesis for the reassembly of PSII [33, 34]. Thus, carotenoid content changes in our experiment evidence in favor of variant 4 being optimal for plants.
Thus, the nanoparticle effect depends upon the plant species, type of nanoparticles, dose of treatment, and method of processing—double treatment at different times (pre-sowing seed treatment as “preparing” and spraying in the budding stage as “fixating” one) caused the best effect. The situation could be considered as hardening. The metal nanoparticles are likely to mobilize hormesis and sensitization through indirect effects on specific biological adaptive mechanisms. Colloidal solution nanometals in small doses used with a certain time interval can be considered as a low-level stress factor that stimulates allostatic response, causing nonlinear modulator changes of endogenously strengthened processes, contributing to the development of adaptive responses and answers.
This work was supported by the State Agency on Science, Innovations and Informatization of Ukraine (according to agreement No. ДЗ/493-2011, 29 09. 2011).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Karuppanapandian T, Moon J-C, Kim C et al (2011) Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms. Aust J Crop Sci 5:709–725Google Scholar
- Prylutskyy YI, Yashchuk VM, Kushnir KM, Golub AA, Kudrenko VA, Prylutska SV et al (2003) Biophysical studies of fullerene-based composite for bio-nanotechnology. Mater Sci Engineer C 23:109–111View ArticleGoogle Scholar
- Prylutska SV, Matyshevska OP, Grynyuk II, Prylutskyy YI, Ritter U, Scharff P (2007) Biological effects of C60 fullerenes in vitro and in a model system. Mol Cryst Liq Cryst 468:265–274View ArticleGoogle Scholar
- Golub A, Matyshevska O, Prylutska S, Sysoyev V, Ped L, Kudrenko V et al (2003) Fullerenes immobilized at silica surface: topology, structure and bioactivity. J Mol Liq 105:141–147View ArticleGoogle Scholar
- Scharff P, Ritter U, Matyshevska OP, Prylutska SV, Grynyuk II, Golub AA et al (2008) Therapeutic reactive oxygen generation. Tumori 94:278–283Google Scholar
- Nair R, Varghese SH, Nair BG, Maekawa T, Yoshida Y, Kumar DS (2010) Nanoparticulate material delivery to plants. Plant Sci 9:154–163View ArticleGoogle Scholar
- Colvin VL (2003) The potential environmental impact of engineered nanomaterials. Nat Biotechnol 9:1166–1170View ArticleGoogle Scholar
- Sytar O, Novicka N, Taran N, Kalenska S, Ganchurin V (2010) Nanotechnology in modern agriculture. Phys Alive 9:113–116Google Scholar
- Kole C, Kole P, Randunu KM, Choudhary P, Podila R, Ke PC et al (2013) Nanobiotechnology can boost crop production and quality: first evidence from increased plant biomass, fruit yield and phytomedicine content in bitter melon (Momordica charantia). BMC Biotechnol 9:37View ArticleGoogle Scholar
- Du W, Gardea-Torresdey JL, Ji R, Yin Y, Zhu J et al (2015) Physiological and biochemical changes imposed by CeO2 nanoparticles on wheat: a life cycle field study. Environ Sci Technol 49:11884–11893View ArticleGoogle Scholar
- Hong F, Zhou J, Liu C, Yang F, Wu C, Zheng L, Yang P (2005) Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach. Biol Trace Elem Res 105:269–279View ArticleGoogle Scholar
- Volkogon V (2006) Microbial preparations in crop production. Theory and practice. Agrarna nauka, KyivGoogle Scholar
- Wei H, Wang E (2013) Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem Soc Rev 42:6060–6093View ArticleGoogle Scholar
- Prylutska SV, Grynyuk II, Matyshevska OP, Prylutskyy YI, Ritter U, Scharff P (2008) Anti-oxidant properties of C60 fullerenes in vitro. Fullerenes, Nanotubes, Carbon Nanostruct 16:698–705View ArticleGoogle Scholar
- Lei Z, Mingyu S, Xiao W, Chao L, Chunxiang Q, Liang C et al. (2008) Antioxidant stress is promoted by nano-anatase in spinach chloroplasts under UV-B radiation. Biol Trace Elem Res 121:69–79Google Scholar
- Song G, Gao Y, Wu H, Hou W, Zhang C, Ma H (2012) Physiological effect of anatase TiO2 nanoparticles on Lemna minor. Environ Toxicol Chem 31:2147–2152View ArticleGoogle Scholar
- Pradhan S, Patra P, Das S, Goswami A (2013) Photochemical modulation of biosafe manganese nanoparticles on Vigna radiata: a detailed molecular, biochemical, and biophysical study. Environ Sci Technol 47:13122–13131View ArticleGoogle Scholar
- Lopatko K, Aftandilyants Y, Kalenska S, Tonkha O (2009) The method for obtaining the solution of non-ionic colloidal metals, Patent for invention №38459. Registered in the State Register of Ukraine patents for utility models., p 12:01Google Scholar
- Giannopolitis CN, Ries SK (1972) Superoxide dismutase I. Occurrence in higher plants. Plant Physiol 59:309–314View ArticleGoogle Scholar
- Aeby H (1984) Catalase in vitro. Methods Enzymol 105:121–126View ArticleGoogle Scholar
- Kumar GNM, Knowles NR (1993) Changes in lipid peroxidation and lipolitic and free-radical scavenging enzyme activities during aging and sprouting of potato (Solanum tuberosum) seed-tubers. Plant Physiol 102:115–124View ArticleGoogle Scholar
- Chevari IS, Andyal T, Strenger J (1991) Determination of antioxidant blood products and diagnostic value in the elderly. Laboratory Work 10:9–13Google Scholar
- Bradford MM (1976) A rapid and sensitive methods for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254View ArticleGoogle Scholar
- Sharma P, Bhatt D, Zaidi MGH, Saradhi PP, Khanna PK, Arora S (2012) Silver nanoparticle-mediated enhancement ingrowth and antioxidant status of Brassica juncea. Appl Biochem Biotechnol 167:2225–2233View ArticleGoogle Scholar
- Salama HMH (2012) Effects of silver nanoparticles in some crop plants, common bean (Phaseolus vulgaris L.) and corn (Zea mays L.). Int Res J Biotechnol 3:190–197Google Scholar
- Ghafariyan MH, Malakouti MJ, Dadpour MR, Stroeve P, Mahmoudi M (2013) Effects of magnetite nanoparticles on soybean chlorophyll. Environ Sci Technol 47:10645–10652Google Scholar
- Racuciu M, Miclaus S, Creanga D (2009) The response of plant tissues to magnetic fluid and electromagnetic exposure. Rom J Biophys 19:73–82Google Scholar
- Green BR, Durnford DG (1996) The chlorophyll-carotenoid proteins of oxygenic photosynthesis. Annu Rev Plant Physiol Plant Mol Biol 47:685–714View ArticleGoogle Scholar
- Maina JN, Wang Q (2015) Seasonal response of chlorophyll a/b ratio to stress in a typical desert species: Haloxylon ammodendron. Arid Land Res Manage 29:321–334View ArticleGoogle Scholar
- Zengin FK, Munzuroglu O (2005) Effects of some heavy metals on content of chlorophyll, proline and some antioxidant chemicals in bean (Phaseolus vulgaris L.) seedlings. Acta Biologica Cracoviensia Series Botanica 47/2:157–164Google Scholar
- Delfine S, Alvino A, Villani MC, Loreto F (1999) Restrictions to carbon dioxide conductance and photosynthesis in spinach leaves recovering from salt stress. Plant Physiol 119:1101–1106View ArticleGoogle Scholar
- Pearcy RW, Sims DA (1994) Photosynthetic acclimation to changing light environments: scaling from the leaf to the whole plant. In: Caldwell MM, Pearcy RW (eds) Ecophysiological processes above and below ground. Academic, New York, pp 145–174Google Scholar
- Depka B, Jahans P, Trebst A (1998) β-Carotene to zeaxanthin conversion in the rapid turnover of the D1 protein of photosystems. FEBS Lett 424:267–270View ArticleGoogle Scholar
- Paulsen H (1997) Pigment ligation to proteins of the photosynthetic apparatus in higher plants. Physiol Plant 100:760–768View ArticleGoogle Scholar