ISSN 00124966, Doklady Biological Sciences, 2013, Vol. 452, pp. 287–290. © Pleiades Publishing, Ltd., 2013. Original Russian Text © Yu.Yu. Nevmerzhitskaya, O.A. Timofeeva, A.L. Mikhaylov, A.S. Strobykina, I.Yu. Strobykina, V.F. Mironov, 2013, published in Doklady Akademii Nauk, 2013, Vol. 452, No. 3, pp. 346–349.

GENERAL BIOLOGY

Stevioside Increases the Resistance of Winter Wheat to Low Temperatures and Heavy Metals Yu. Yu. Nevmerzhitskayaa, O. A. Timofeevaa, A. L. Mikhaylova, A. S. Strobykinaa, I. Yu. Strobykinab, and Corresponding Member of the RAS V. F. Mironovb Received June 5, 2013

DOI: 10.1134/S0012496613050098

One of the main tasks of modern agricultural sci ence is the identification and development of new plant growth regulators that are environmentally safe, multifunctional, physiologically active compounds with both growthregulating and antistress proper ties. Steviol (13hydroxykaur16en18oic acid), the aglycon of diterpene glycosides of the plant Stevia rebaudiana Bertoni, is of particular interest [1]. In the literature, there is evidence that derivatives of steviol glycosides show gibberellinlike activity [2]. The use of plant growth and development regulators that reduce the toxicity of pollutants and the accumulations of pollutants in plants is very important under the condi tions of increasing heavy metal pollution of agricul tural lands. In this connection, the goal of this work was to determine the protective effect of the giberellin like diterpenoid glycoside stevioside on winter wheat under the influence of low temperatures and heavy metals. The results of the research have demonstrated that the diterpene glycoside stevioside (10–8 M) increases the cold resistance of winter wheat to a higher extent in comparison with its derivatives obtained by chemi cal means and reduces the effects of cadmium and zinc on plant growth and changes of lectin activity. We studied the roots of seedlings of the winter wheat (Triticum aestivum L.) cultivar Mironovskaya 808. The studied compounds were synthesized in the Arbuzov Institute of Organic and Physical Chemistry of the Kazan Scientific Center of the Russian Acad emy of Sciences (Kazan). Plants were grown under laboratory conditions in cuvettes with tap water at a light intensity of 100 W/m2 and a 12h photoperiod at

23°C for nine days. The treated plants were grown on a stevioside (10–8 M) solution. Then, fiveday old plants were transferred to heavy metals solutions of CdSO4 and ZnSO4 at concentrations of 10 µM and 1 mM. The concentrations of stevioside and heavy metals were selected in preliminary experiments. Sol uble lectins were extracted with 0.05 N HC1, cellwall lectins were extracted with 0.05% Triton X100. The lectin activity was estimated by hemagglutination with erythrocytes of blood group O [3]. Protein content was determined accordingly to Bradford [4]. Cold resis tance was tested based on electrolytes leakage [5]. All experiments were performed in three biological replicates. The results shown in the figures and tables are arithmetic means and their standard errors. At the first stage of the research, we identified the compound with the highest antistress activity in winter wheat. For this identification, LT50 were determined in seedlings grown on solutions of (1) stevioside 10–8 M and its derivatives: (2) steviol (10–8 M), (3) dihydroste viol (10–8 M), (4) steviolbioside (10–8 M), and (5) bis dihydrosteviol malonate (10–8 M). As can be seen in Table 1, all studied compounds increased the cold tolerance of plants in comparison with the control. The effect of stevioside derivatives on the cold resistance of seedlings is comparable to that of absci Table 1. The influence of stevioside and its derivatives on LT50 LT50, °C

Variant Н2О

–6.2 ± 0.2

Stevioside (10–8 M) –8

Steviol (10

a

Department of Plant Physiology and Biochemistry, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, 420008 Tatarstan, Russia b Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences, Kazan, 420008 Tatarstan, Russia

–8.0 ± 0.2

M)

–7.4 ± 0.1

Digidrosteviol (10–8 M)

–6.9 ± 0.3

Steviolbioside (10–8 M)

–7.3 ± 0.1 –8 M)

Bisdihydrosteviol malonate (10 287

–7.5 ± 0.1

288

NEVMERZHITSKAYA et al.

Table 2. Effect of stevioside on the length of the roots and the first true leaves of ninedayold seedlings of winter wheat Mironovskaya 808 Н2О

Variant

Stevioside

leaf length, mm

root length, mm

leaf length, mm

root length, mm

159 ± 2.0 69 ± 2.5 129 ± 1.5 133 ± 2.7 157 ± 3.5

90 ± 2.3 45 ± 3.8 64 ± 2.5 73 ± 1.5 76 ± 1.5

180 ± 2.1 100 ± 3.5 134 ± 3.0 141 ± 2.3 172 ± 1.8

107 ± 3.3 67 ± 2.7 71 ± 1.7 87 ± 2.5 98 ± 3.1

Control CdSO4 (1 mM) CdSO4 (10 µM) ZnSO4 (1 mM) ZnSO4 (10 µM)

sic acid (ABA) (ABA decreased the LT50 of wheat seed lings to ⎯7.6°C) [6], and stevioside effect was even slightly higher than that of ABA (LT50 of wheat seedlings as low as 8°C). OH

Cultivation of winter wheat on a stevioside solution (10–8 M) caused an increase in the leaf length by 14% and root length by 18% in comparison with the control (Table 2).

OH OH

HO

HO O

HO

O

O

OH

O

OH

OH

OH O

CH2

OH

H

O O

OH COOH

OH 1

2

OH HO

COOH 3

OH OH

HO O

O

O

HO

O

O

O

O

O

OH

HO

COOH 4

Both high (1 mM) and low (10 µM) concentrations of ZnSO4 and CdSO4 caused a decrease in the leaf and root lengths of the wheat seedlings (Table 2). Growth inhibition is a common manifestation of heavy metal toxicity for plants; this inhibition is mainly due to the

O

O

OH

5

direct effect of heavy metals on cell division and extension [7]. Cadmium has the most toxic effect on wheat growth. Different authors have demonstrated that toxic heavy metal concentrations vary consider ably [8], which, apparently, is due to the physiological DOKLADY BIOLOGICAL SCIENCES

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STEVIOSIDE INCREASES THE RESISTANCE OF WINTER WHEAT −Stevioside

+Stevioside

300 Lectin activity, % of control

Lectin activity, % of control

120 100 80 60 40 20 0

1 2

3 4

1 2

3 4

Fig. 1. Effect of CdSO4 and ZnSO4 on lectin activity of the cell wall in the pretreated with stevioside (10–8 M) (+ Stevi oside), and untreated (– Stevioside) roots of seedling of win ter wheat cultivar Mironovskaya 808: (1) CdSO4 (1 mM); (2) ZnSO4 (1 mM); (3) CdSO4 (10 µM); (4) ZnSO4 (10 µM).

and agerelated characteristics of the object of study, the selected experimental methodology, composition and acidity of the environment, and other factors. [9] Modification of the effect of heavy metals on crop plants by using various growth regulators was shown in several studies [10]. Pretreatment of the winter wheat cultivar Mironovskaya 808 with stevioside for five days reduced the inhibitory effects of both cadmium sulfate and zinc sulfate on seedling growth (Table 2). Growth regulators primarily act via changes in the synthesis and activity of various proteins. In literature, there are data indicating the presence of phytohor mone binding sites other than carbohydrate binding sites in plant lectin molecules. For example, wheat germ agglutinin (WGA) has a high affinity for auxins, cytokinins, and gibberellic acid [11]. The researchers assume that the lectin–phytohormone complex not only serves as a transport form of hormones, but also may be involved in the regulation of plant growth and development [11]. Therefore, we determined the lec tin activity in plants grown at medium contents of heavy metals and stevioside (10–8 M) (Figs. 1, 2). Heavy metals decreased the activity of lectins of the cell wall (Fig. 1). These changes may be due to both the adsorption of heavy metals, as in the root cell walls [7], and the interaction of metal ions with sulfhydryl groups of proteins, leading to the inhibition of their activity and/or alteration of the structure [12]. At the same time, CdSO4 and ZnSO4 significantly increased the activity of soluble lectins (Fig. 2), mainly, WGA. An increased WGA content [13] accompanied by an increase in the release of the lectin to the environment was detected in the roots of wheat seedlings exposed to cadmium acetate. DOKLADY BIOLOGICAL SCIENCES

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−Stevioside

+Stevioside

1 2

1 2

250 200 150 100 50 0

3 4

3 4

Fig. 2. Effect of CdSO4 and ZnSO4 on the activity of solu ble lectins in the in the pretreated with stevioside (10–8 M) (+ Stevioside), and untreated (– Stevioside) roots of seed ling of winter wheat cultivar Mironovskaya 808.

After pretreatment of plants for five days with stevi oside (10–8 M), the effect of heavy metals on the lectin activity of the cell wall was similar to the changes in the growth parameters: in the presence of stevioside, the susceptibility of cell wall lectins to CdSO4 and ZnSO4 decreased (Fig. 1). The activity of soluble lectins upon joint action of stevioside and heavy metals was signifi cantly suppressed (Fig. 2). At present, hardly any information on the mechanisms of the action of stevi oside is available; however, experiments with animals have demonstrated that this glycoside is an antagonist of calcium and reduces the permeability of calcium channels [14]. It can be assumed that by this mecha nism stevioside modifies Cd and Zn absorption and transport in root cells. Thus, according to the study, stevioside (10–8 M) reduces the effect of cadmium and zinc on plant growth and changes the activity of lectins, indicating that it has a protective effect on winter wheat under the stress caused by heavy metals. REFERENCES 1. Kataev, V.E., Khaibullin, R.N., Sharipova, R.R., and Strobykina, I.Yu., Obzorn. Zh. Khim., 2011, vol. 1, no. 1, pp. 99–167. 2. Timofeeva, O.A., Nevmerzhitskaya, Yu.Yu., Mifta khova, I.G., Strobykina, A.S., Mikhailov, A.L., Stro bykina, I.Yu., and Mironov, V.F., Dokl. Biol. Sci., 2010, vol. 435, no. 2, pp. 411–414. 3. Timofeeva, O.A., Lectins as components of adaptive responses of winter wheat to adverse environment, Extended Abstract of Doctoral (Biol.) Dissertation, Ufa, 2009. 4. Bradford, M.A., Biochemistry, 1976, vol. 72, pp. 248– 254. 5. Uemura, M. and Steponkus, P.L., Plant Physiol., 1989, vol. 91, no. 3, pp. 961–969.

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6. Khokhlova, L.P., Olinevich, O.A., Tarakanova, N.Yu., Timofeeva, O.A., Volovnik, I.L., Palikh, E., and Rau daskoski, M., in Grani sotrudnichestva: K 10letiyu Soglasheniya o sotrudnichestve mezhdu Kazanskim i Gis senskim universitetami: Sbornik nauchnykh statei i obzornykh materialov (Aspects of Collaboration: On the 10th Anniversary of the Agreement on Collaboration between the Universities of Kazan and Giessen: Collec tion of Research Articles and Reviews), Kazan: Uni press, 1999, pp. 275–298. 7. Titov, A.F., Talanova, V.V., Kaznina, N.M., and Laid inen, G.F., Ustoichivost’ rastenii k tyazhelym metallam (Plant Resistance to Heavy Metals), Petrozavodsk: Karel’skii Nauchnyi Tsentr RAN, 2007. 8. Kopittke, P.M., Blamey, F.P.C., Asher, C.J., and Men zies, N.W., J. Exp. Bot., 2010, vol. 61, no. 4, pp. 945– 954.

9. Kulikova, A.L., Kuznetsova, N.A., and Kholodova, V.P., Fiziol. Rast., 2011, vol. 58, no. 5, pp. 719–727. 10. Bashmakov, D.I., Pynenkova, N.A., Sazanova, K.A., and Lukatkin, A.S., Fiziol. Rast., 2012, vol. 59, no. 1, pp. 67–73. 11. Bogoeva, V.P., Radeva, M.A., Atanasova, L.Y., Stoits ova, L.Y., and Boteva, R.N., Biochim. Biophys. Acta, 2004, vol. 1698, no. 2, pp. 213–218. 12. Van Assche, F. and Clijsters, H., Plant, Cell Environ., 1990, vol. 13, pp. 195–206. 13. Bezrukova, M.V., Fatkhutdinova, R.A., Lubya nova, A.R., Murzabaev, A.R., Fedyaev, V.V., and Sha kirova, F.M., Fiziol. Rast., 2011, vol. 58, no. 6, pp. 907– 914. 14. Melis, M.S., Braz. J. Med. Biol. Res., 1992, vol. 25, no. 9, pp. 943–949.

Translated by V. Mittova

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2013