ADAPTATION REACTIONS OF COMMON WHEAT (TRITICUM AESTIVUM L.) AND EMMER (T. DICOCCUM SCHRANK EX SCHÜBL.) SEEDLINGS UNDER OSMOTIC STRESS AND TREATMENT WITH METAL NANOPARTICLES

ADAPTATION REACTIONS OF COMMON WHEAT (TRITICUM AESTIVUM L.) AND EMMER (T. DICOCCUM SCHRANK EX SCHÜBL.) SEEDLINGS UNDER OSMOTIC STRESS AND TREATMENT WITH METAL NANOPARTICLES M. M. Musienko 1, Ya. M. Gadzalo 2, M. S. Kovalenko 1, L. M. Batsmanova 1, Ye. O. Konotop 1, N. Yu. Taran 1 1 Taras Shevchenko National University of Kyiv 64/13, Volodymyrska Str., Kyiv, 01601, Ukraine 2 National Academy of Agrarian Sciences, 9, Omelianovych-Pavlenko Str., Kyiv, 01010, Ukraine


INTRODUCTION
The search for the means of adapting wheat to drought due to global climate changes to ensure food safety is a complicated task for scientists, requiring additional investigations [1][2][3]. International researchers work at solving this problem, elaborating new approaches to enhance cereal productivity using modern methods of agricultural [4] and breeding technologies [5][6][7][8].
The most relevant requirement, which should be met by the perspective cultivars, is adaptivity, i.e. the capability of resisting environmental factors, decreasing productivity and yield. Non-compliance with adaptivity requirements leads to a rise in price for agricultural products [9]. Expert community of Wheat Yield Consortium (WYC) are of the opinion that slower tempo of the increase in productivity is related to exhausted possibilities of enhancing it due to the factors, which ensured the thrust in wheat productivity rise as a result of "green revolution" [10]. At the same time, being one of the leading food crops, wheat may have suffered the most from so called "genetic erosion" [11]. At present only two cultivars -common wheat Triticum aestivum L. and, to a lesser degree, durum wheat T. durum Desf. cover practically the whole area of the crop [12]. The reduction in the genetic make-up, and thus in the diversity of genes, conditioning the resistance to biotic and abiotic factors makes fi elds susceptible, and the volume and quality of the yield -unstable [7,13].
Recent decades have witnessed the renewed interest to non-traditional species of wheat, fi rst and foremost, spelt (Triticum spelta L.) [14,15] and emmer (T. dicoccum Schrank ex Schübl.) [13,16]. A common feature of these species is their grain being tightly enclosed with husks, which makes it hard to thresh -hull content [12]. Hull presence limits the cultivation of these species, as it complicates their processing with the purpose of obtaining pure grain, which requires special equipment and additional energy losses. However, husks are reliable protection for the grain kernel and young seedling during the period from sowing to sprouting [17]. The resistance of young and mature plants to pests and diseases is also ensured by the resistance genes [18], which allows avoiding chemical means of protecting plants and meets the requirements of organic agriculture [12].
Drought and increased temperatures have negative effect on the growth and development of plants, water regime, induce shorter ontogenesis stages, disrupt photosynthesis processes (light absorption, fi xation of CO 2 ) and respiration which leads to the loss of cereal productivity [19,20]. A cascade of chain reactions is developed on the cellular level, which conditions the disruption of redox homeostasis and, as a result, the increase in the production of reactive oxygen species (ROS) [21]. The induction of different protection mechanisms occurs in plants under these conditions: elimination of ROS, synthesis of antioxidants, accumulation of osmotic active substances, activation of signaling cascades [22,23] which lead to the formation of different strategies of adaptation to drought.
Therefore, global warming and droughts trigger general concern for the production of grain and other crop products. The solution to this problem is impossible without accumulating new knowledge about metabolome self-regulation mechanisms under drought conditions [24], in cereal crops, in particular. The study of plant adaptation to drought is described in numerous articles in global scientifi c literature, but so far, none of the investigations led to desired enhancing of plant tolerance to osmotic stress, which is evidently related to insuffi cient knowledge of physiological and biochemical foundations of forming adaptive reactions under these conditions.
One of the mechanisms of compensating negative impact of drought is the application of mineral nutrition elements [25]. In this respect, a promising method is the application of nanofertilizers in the form of nonionic colloid metal nanoparticles, but the introduction of such methods requires thorough study of their impact on biological objects. It is believed that nanoparticles of essential metals are involved in different metabolitic processes, including photosynthesis, respiration and assimilation of nitrates, which is conditioned by their cofactor function [26]. In particular, Cu, Fe, Mn and Zn nanoparticles are likely to modulate synthesis and functions of antioxidant enzymes [27]. Thus, one of the mechanisms of stress-protective activity of metal nanoparticles may be the regulation of ROS level and functioning of cellular antioxidant system. The aim of this work was to study adaptive reactions of common wheat and emmer under polyethylene glycol (PEG)-induced osmotic stress and treatment with the colloid solution of metal (Fe, Cu, Mn, Zn) mixture nanoparticles. to study the specifi cities of defensive reactions of different genotypes under osmotic stress. The roots and leaves of 7-day-old seedlings were used in the study. The plants were grown on Hoagland's solution with the osmotic potential of -0.3 MPa. The negative osmotic potential in the medium was created using PEG 6000 (Carl Roth, Germany) according to Michel and Kaufmann [28]. PEG was not added to control plants.

MATERIALS AND METHODS
To investigate the possibility of eliminating the negative impact of osmotic stress on seedlings, the seeds of the investigated cultivars were subject to presowing treatment via processing with the colloid solution of metal nanoparticles mixture (Fe, Cu, Mn, Zn) in the concentration of 2 mg/l for 12 h. The colloid solutions of metal nanoparticles were elaborated by the Chair of technology of construction materials and materials science, NULES of Ukraine, and obtained via dispersion of granules of the relevant metals with electric impulses of 100-2,000 A in water [29].
The development of oxidative processes was estimated by the content of thiobarbituric acid (TBA) reactive substances [30]. Plant tissues (200 mg) were homogenized with 3 ml 0.1 M buffer of Tris-HCl (pH 7.6). The homogenate was added 1 ml of 0.67 % TBA solution and 2 ml of 20 % trichloroacetic acid. The reaction mixture was boiled on water bath for 30 min and centrifuged at 1,500 g for 10 min. The absorption was defi ned at λ 533 nm using spectrophotometer Shimadzu UV-1500 (Japan). The content of TBA-reactive substances was estimated using the coeffi cient of molar extinction for malondialdehyde (= 1.55·10 5 M -1 ·cm -1 ) and expressed in μmol per g of fresh weight.
The content of proline was determined according to the method of Bates et al. (1973) [31]. For this purpose, the plant material (0.15 g) was homogenized in 2 ml of 3 % solution of sulfosalicylic acid and centrifuged at 7,000 g for 10 min. The reaction mixture, containing 1 ml of supernatant, 1 ml of acid ninhydrin solution (1.25 g ninhydrin in 60 % acetic acid and 40 % 6М Н 3 РО 4 ) and 1 ml of iced acetic acid, was incubated on boiling water bath for 60 min. After cooling, the tubes were added 3 ml of benzene, mixed and kept at room temperature for 30 min until the separation of two phases. The absorbance of solutions (benzene fraction) was measured at  520 nm to the pure benzene. The estimation of proline content in the investigated samples was conducted by the calibration curve and expressed in μg per g of fresh weight.
To determine the activity of antioxidant enzymes, plant tissues were homogenized in liquid nitrogen with the ad-dition of 50 mM potassium-phosphate buffer (pH 7.8). The obtained homogenate was centrifuged at 12,000 g for 15 min at 4 °C. The activity of superoxide dismutase (SOD, EC 1.15.1.1) was measured according to Giannopolitis and Ries (1977) [32]. For this purpose, 50 ml of the extract were added to the reaction mixture, containing 1 ml of 0.0015 % ribofl avin, 1 ml of 5.82 % methionine, and 1 ml of 0.154 % of nitroblue tetrazolium. The absorption of the solution was estimated at  560 nm after the incubation of the reaction mixture in light for 15 min. SOD activity was estimated as the amount of the enzyme, inhibiting the rate of formazan formation by 50 %, and expressed in conditional units per mg of protein.
The studies were conducted in three biological and three analytical repeats. The statistical analysis of the data obtained was conducted in Microsoft Excel and Statistica 8.0. The comparison of samples involved the use of arithmetic mean (M) and standard error of mean (SEM). The reliability of the difference between the compared groups was estimated using Duncan's criterion. The difference p < 0.05 was considered statistically reliable for all the indices.

RESULTS
The investigation of the content of TBA-reactive substances in the roots and leaves of common wheat and emmer demonstrated that the development of lipid peroxidation under osmotic stress was observed only in the seedlings of cv. Trypilska (Fig. 1). For instance, the content of TBA-reactive substances in the roots of seedlings increased by 60 % compared to the control variant, and in the leaves -by 27 %. The results obtained demonstrated the changes in proline metabolism in the investigated species under the stress. It was established that proline content in different parts of common wheat seedlings is different, with considerable prevalence (10 : 1) in the roots (Fig. 2). Less intense accumulation of proline was observed in the roots of emmer seedlings, cv. Holikovska, compared to other cultivars. At the same time, no reliable signifi cant changes in proline content were determined in the leaves of common wheat seedlings, contrary to emmer, where this index was 7 times higher.
After the presowing treatment with the mixture of metal nanoparticles, the decrease in proline content was observed in the roots and leaves of emmer under osmotic stress by 66 and 36 % respectively, compared to the variant, not treated with nanoparticles. The established changes may demonstrate the involvement of alternative protective mechanisms, in particular, the systems of antioxidant protection.
The investigation of the activity of the main antioxidant enzymes, including SOD and CAT, established the multi-vector defensive reactions of the investigated species under prolonged osmotic stress. An increase of SOD activity by 30 % under osmotic stress was shown in roots of common wheat of cv. Favorytka, while it decreased by 25 % in the roots of cv. Trypilska (Fig. 3). At the same time, the level of this enzyme activity in the roots of emmer of cv. Holikovska corresponded to control values. SOD activity in the leaves of the studied cultivars under stress conditions was not changed.   Drought-induced proline accumulation is known to be one of adaptive reactions of many cereal crops [35]. The results of our investigations demonstrate that osmotic regulation and maintenance of water balance of cells under stress takes place with some participation of proline, which is also confi rmed with considerable accumulation of this aminoacid in the roots of the investigated cultivars. The root system has direct contact with the medium of negative osmotic potential, and thus should ensure the conditions to preserve the water content, suffi cient for metabolic processes, and make water outfl ow into the medium impossible. In this respect, the accumulation of osmotically active proline in the roots is an expected reaction of plants under osmotic stress [36].
According to the scientifi c literature, proline accumulation in the leaves may result from changes in plant metabolism under drought, aimed at the decomposition and decrease in protein synthesis or transformation of some aminoacids into proline [37]. The role of proline in plant leaves under stress is not limited with osmotic regulation only. Proline is known to act as chaperon, ensuring the protection of protein molecules, has antioxidant properties and participates in the regulation of nitrogen content [38]. At the same time, the mechanism of maintaining osmotic balance depends on the genotype. In particular, the capability of accumulating proline in response to drought may serve as a criterion to enhance drought-resistance of wheat in the breeding programs (for instance, the selection of genotypes with higher content of proline under drought conditions compared to normal conditions) [39].
Our results are in good agreement with, for instance, the investigation of Marcińska et al. [40] where a 2-5-fold increase in proline content was registered in the leaves of common wheat seedlings (T. aestivum) depending on the PEG content in the cultivation medium. The authors determined that the accumulation of this aminoacid occurred more intensely in cultivars with higher drought-resistance compared to drought-sensitive ones. It is known that exogenous treatment with proline may decrease the negative effect of stress, in particular, pollution with heavy metals, and promote avoiding excessive moisture loss by plants [41].
Osmotic stress is usually accompanied with the increase in ROS generation and activation of the antioxidant system of plants. The rate of changes in the content and activity of its components and the capability to maintain redox-balance at the level, which allows avoiding critical oxidative damage of cells, is required to ensure the survival of plants under unfavorable environmental factors. For instance, the study of Zhang and Kirkham [42] demonstrated that under drought similar SOD and CAT reaction is observed in 10-day-old wheat seedlings with different genome ploidy. At the same time, the authors indicate lower effi ciency of antioxidant systems of common wheat compared to emmer, depending on the degree of lipid peroxidation. The study of Sairam, R. K. et al. [43] demonstrate higher SOD activity and lower content H 2 O 2 and malondialdehyde in emmer under drought on different stages of ontogenesis which also demonstrates more profound mechanism of antioxidant protection of plants of this species. It is noteworthy that usually stress is induced in seedlings/mature plants, previously grown under optimal moisturization conditions. The osmotic stress system, used in our study, allows estimating the condition of plants, which grew under osmotic stress since germination.
Little is still known about the impact of metal nanoparticles on different levels of antioxidant protection of plants. Our results demonstrate the protective effect from the treatment with metal nanoparticles mixture, observed in the roots of the investigated species, where a decrease in the content of TBA-reactive substances took place on the background of the increase in SOD and CAT activity. It is believed that the treatment with metal nanoparticles may mediate the activation of the system of antioxidant protection, promoting rapid adaptation of plans under stress conditions [26,44]. Metal nanoparticles may be the source of ROS and promote the intensifi cation of reactions within the antioxidant system [45,46]. Therefore, they may facilitate pre-adaptation of plants to the effect of unfavorable environmental factors [47] which is in agreement with our results. Moreover, some metal oxide nanoparticles manifest enzyme-like activity, in particular, these are nanoparticles of Fe, Cu and Mn oxides [45]. Taking into consideration the fact that SOD cofactors are Cu, Fe, Mn, Zn, and CAT contains heme Fe [48], nanoparticles of the relevant metals could be involved in the composition of antioxidant enzymes. Some investigations demonstrate that the treatment with nanoparticles of Cu oxide promotes the increase in SOD activity in duckwheat plants (Lemna minor L.) [49], rice (Oryza AGRICULTURAL SCIENCE AND PRACTICE Vol. 6 No. 3 2019 sativa L.) [50] and Arabidopsis (Arabidopsis thaliana (L.) Heynh.) [51]. Wang et al. demonstrated that after treatment with nanoparticles of Fe oxide, there is an increase in SOD and CAT activity in the plants of perennial ryegrass (Lolium perenne L.) and pumpkin (Cucurbita mixta) [52]. Later Hu et al. observed a similar reaction after treatment with nanoparticles of Zn oxide in salvinia plants (Salvinia natans (L.) All.) [53]. At the same time, there is no confi rmation of changes in enzyme activity, conditioned by their direct interaction with nanoparticles [45].
Regardless of a great number of studies, the issue of phytotoxicity of nanoparticles is still under discussion, in particular, due to their application in toxicological tests in extremely high concentrations [54]. Some studies demonstrate dose-dependent phytotoxic effect of applying nanoparticles of metals and their oxides [50,51]. At the same time, the application of nanoparticles in lower concentrations may promote the growth, development of plants and mitigate the manifestations of abiotic stresses of different origin. For instance, the treatment with Fe nanoparticles promotes the decrease in negative effect of drought on saffl ower plants (Carthamus tinctorius L.) [55], Arabidopsis (A. thaliana) [56] and wheat (T. aestivum L.) [57]. Nanoparticles of Cu and Zn oxides may also manifest protective effect under drought, in particular, in wheat plants [58]. At present, there are scarce data about the effect of metal nanoparticles mixtures on plants. Thus, the results of our studies may become the basis for further study on complex effect of nanometals. Therefore, the determination and selection of drought-resistant genotypes remains a problem, as the development of adaptive reactions of common wheat and emmer seedlings under polyethylene glycol (PEG)-induced osmotic stress is considerably species-and cultivar-specifi c. For instance, the reaction of drought-resistant cultivar seedlings of common wheat Favorytka and that of emmer, cv. Holikovska, to the stress were different. The activation of the system of antioxidant protection in drought-resistant genotypes usually occurs in coordination or synergy to prevent the damage of cells, which promotes the increase in drought resistance.