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Influence of stevioside and heavy metals on physiological and biochemical parameters of winter wheat.


Secondary metabolites of plants constantly cause researchers' interest due to their various biological activity and ability to serve as base for creation of plants growth regulators. Among secondary metabolites, diterpene glycosides of Stevia rebaudiana Bertoni plants with steviol (13-hydroxy-ent-kaur-16-en-19-oic acid) as aglycon is of particular interest [1]. Previously, steviol was considered as precursor of gibberellic acid due to cis-coupling of B and C rings of tetracyclic hydrocarbon system, typical for gibberellins. However, it was discovered that Gibberelle fujikuroi fungus does not turn steviol into gibberellic acid, but metabolize it to gibberellins-like compound [2]. There is also data in literature about steviol glycoside derivatives that show gibberellins-like activity [3]. Earlier, it was shown that main stevia glycoside--stevioside--has antistress properties and increases frost resistance of wheat plants [4]. The aim of this study was to discover protective abilities of gibberellins-like diterpene stevioside glycoside on winter wheat plants under heavy metals influence.


Objects of the study were roots of winter wheat germinating seedlings (Triticum aestivum L., Mironovskaya 808 type). Diterpene stevioside glycoside was obtained from raw Stevia rebaudiana Bertoni plant in the Laboratory of phosphorus analogs of natural compounds (Head of the Laboratory is Doctor of Chemistry, Professor, the Corresponding Member of RAS Vladimir F. Mironov) in AE Arbuzov Institute of Organic and Physical Chemistry. Plants were grown in the laboratory in sample chambers with tap water under the conditions of light at 100 W/[m.sup.2] and 12-h photoperiod at temperature 23[degrees]C for 9 days. In experimental samples, the plants were grown on diterpene stevioside glycoside solution ([10.sup.-8] mole) for 5 days. After 5 days, plants have been transferred to heavy metals solutions (CdS[O.sub.4], CuS[O.sub.4], and ZnS[O.sub.4]) in 10 [micro]mol concentration. Concentration of stevioside and heavy metals was chosen according to preliminary experiments. Amounts of heavy metals in wheat germinating seedlings were determined in mass spectrometer Elan DRC II (PerkinElmer, USA) [5]. Activity of ascorbate peroxidase was determined by the method described in Ref. [6]. Extraction and defining of proline were conducted by the method described in Ref. [7]. Experiments were performed in biological triplicate.

Results and Discussion

Growing of winter wheat plants on stevioside solution ([10.sup.-8] mole) caused the increase the height of germinating seedlings leaves by 14% and length of the roots by 18% in comparison to the control samples (Table 1). It is reported in literature that some diterpenoids and glycosides of ent-Kaurane Glycosides boost the plants growth [3].

CdS[O.sub.4], CuS[O.sub.4], and ZnS[O.sub.4] inhibited the growth of germinating seedlings roots and leaves in different measures (Table 1). Decrease in growth speed is a common effect of heavy metals on plants, based on their direct influence on division and stretching of cells [8,9]. As it is shown in Table 2, a significant amount of [Cd.sup.2+], [Cu.sup.2+], [Zn.sup.2+] has been accumulated in germinating seedlings roots and leaves under these conditions (Table 2). Modification of heavy metals influence on cultivated plants pacreHHR by applying different growth regulators is shown in Ref. [10]. In our experiments, stevioside decreased heavy metals influence on the plant growth (Table 1) and lowered heavy metals accumulation level in germinating seedlings (Table 2).

There is a number of data about heavy metals influence on active oxygen generation in literature [11-13]. Antioxidant enzymes, including a wide group of peroxidases, take part in detoxification of active oxygen. Ascorbate peroxidase is a main enzyme involved in detoxification of [H.sub.2][O.sub.2] in the cell, due to oxidation of ascorbic acid. [Cd.sup.2+] and [Zn.sup.2+] inhibited activity of this enzyme, but [Cu.sup.2+] increased ascorbate peroxidase activity by 50% (Figure 1). Pretreatment of germinating seedlings with stevioside decreased the effect of [Cu.sup.2+] on ascorbate peroxidase activity to the level of control plants, while ascorbate peroxidase activity increased by 38% and 56% under the influence of [Cd.sup.2+] and [Zn.sup.2+], respectively, in comparison to control plants (Figure 1).

Despite the important role of antioxidant enzymes in various types of stress, low-molecular organic antioxidants can protect metabolism from active oxygen more effectively in some cases [14]. One of these compounds is proline amino acid.

According to the obtained data, addition of heavy metals into plants growing medium significantly increases the proline level. Proline accumulates the most with [Cu.sup.2+]. Some scientists connect heavy metals resistance with proline accumulation [15].

Stevioside stimulated proline accumulation in germinating seedlings roots up to 4 times in comparison to plants grown on water. We suggest that in this case the proline level may indicate the increase of adaptive potential of stevioside-treated plants. Proline concentration increased even higher in germinating seedlings treated with heavy metals and stevioside in comparison to treatment with pollutants only (Table 3).

At present, there is a very few data on stevioside mechanism of action, but experiments with animals have shown that this glycoside is a calcium channel blocking agent [16]. It can be supposed that stevioside changes adsorption and transportation of heavy metals in plant cells in the same way.

Thus, according to the performed research, stevioside ([10.sup.-8] mol) decreased metals effect of plants growth and lowered their accumulation level in germinating seedlings and also caused a significant growth of proline level, which indicates its protective action on winter wheat plants under heavy metals caused stress.


1. It is determined that CdS[O.sub.4], ZnS[O.sub.4], and CuS[O.sub.4] induced proline accumulation, which indicates that the main protective function under oxidation stress caused by heavy metals is carried out by low-molecular no-enzymatic.

2. Stevioside lowered heavy metals accumulation level in roots and leaves of winter wheat germinating seedlings, decreased pollutants effects on plant growth, increased ascorbate peroxidase activity and proline content, which indicated its protective action on winter wheat plants under heavy metals caused stress.


The work was performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.


[1.] Kovylyaeva GI, Bakaleinik GA, Strobykina IYu, Gubskaya VI, Sharipova RR, et al. (2007) Glycosides from Stevia rebaudiana. Chemistry of Natural Compounds 43(1): 81-85.

[2.] Ruddat M, Heftman E, Lang A (1965) Conversion of steviol to a gibbere llin-like compound by Fusarium moniliforme. Archives of Biochemistry and Boiphysics 111: 187-190.

[3.] Timofeeva OA, Nevmerzhitskaya YY, Miftakhova IG, Strobykina AS, Mikhailov AL, et al. (2010) Diterpenoid steviol derivatives regulate the growth of winter wheat and improve its frost resistance. Doklady Biological Sciences 435(1): 411-414.

[4.] Nevmerzhitskaya YY, Timofeeva OA, Mikhailov AL, Strobykina AS, Strobykina IY, et al. (2013) Stevioside increases the resistance of winter wheat to low temperatures and heavy metals. Doklady Biological Sciences 452(1): 287-290.

[5.] ICP Mass Spectrometry Handbook (2005). Nelms SM (Ed.). Oxford (UK)/Carlton (Australia): Blackwell Publishing Ltd/CRC Press, p. 485.

[6.] Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and Cell Physiology 22: 867-880.

[7.] Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water stress studies. Plant Soil 39: 205-207.

[8.] Yang YY, Jung JY, Song W-Y, Suh H-S, Lee Y (2000) Identification of rice varieties with high tolerance or sensitivity to lead and characterization of the mechanism of tolerance. Plant Physiology 124: 1019-1026.

[9.] Sandalio LM, Dalurzo HC, Gomes M, Romero-Puertas MC, del Rio LA (2001) Cadmium-induced changes in the growth and oxidative metabolism of pea plants. Journal of Experimental Botany 52 (364): 2115-2126.

[10.] Bashmakov DI, Pynenkova NA, Sazanova KA, Lukatkin AS (2012) Effect of the synthetic growth regulator Cytodef and heavy metals on oxidative status in cucumber plants. Russian Journal of Plant Physiology 59(1): 59-64.

[11.] Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inze D, et al. (2000) Dual action of the active oxygen species during plant stress responses. Cellular and Molecular Life Sciences 57: 779-795.

[12.] Mitteler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7(9): 405-410.

[13.] Fang W-C, Kao CH (2000) Enhanced peroxidase activity in rice leaves in response to excess iron, copper, and zinc. Plant Science 158: 71-76.

[14.] Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress: A review. Annals of Botany 91: 179-194.

[15.] Tandon PK, Srivastava M (2004) Effect of cadmium and nickel on metabolism during early stages of growth in gram (Cicer arietinum L.) seeds. Indian Journal of Agricultural Biochemistry 17(1): 31-34.

[16.] Melis MS (1992) Influence of calcium on the blood pressure and renal effects of stevioside. Brazilian Journal of Medical and Biological Research 25(9): 943-949.

Published: 19th Dec 2014

Julia Yurevna Nevmerzhitskaya *, Alexander Leonidovich Mikhailov, Anastasia Sergeyevna Strobykina, Olga Arnoldovna Timofeeva

Kazan (Volga region) Federal University, Kremlevskaya 18, 420008, Kazan, Russia.

* Corresponding author

Table 1: Influence of stevioside on roots length and leaves
height of 9-day-old germinating seedlings of Mironovskaya 808
winter wheat.

Sample                           [H.sub.2]O

                  Leaves length (mm)   Roots length (mm)

Control           158.5 [+ or -] 2.0   90.3 [+ or -] 2.3

CdS[O.sub.4]        129 [+ or -] 1.5     64 [+ or -] 2.5
(10 [micro]mol)

ZnS[O.sub.4]        157 [+ or -] 3.5   75.5 [+ or -] 1.5
(10 [micro]mol)

CuS[O.sub.4]        136 [+ or -] 1.9     51 [+ or -] 1.5
(10 [micro]mol)

Sample                         Stevioside

                       Leaves             Roots
                    length (mm)        length (mm)

Control           180 [+ or -] 2.1   107 [+ or -] 3.3

CdS[O.sub.4]      134 [+ or -] 3.0    71 [+ or -] 1.7
(10 [micro]mol)

ZnS[O.sub.4]      172 [+ or -] 1.8    98 [+ or -] 3.1
(10 [micro]mol)

CuS[O.sub.4]      143 [+ or -] 1.5    71 [+ or -] 3.2
(10 [micro]mol)

Table 2: Influence of stevioside on heavy metals content in 9/
day/old germinating seedlings of Mironovskaya 808 winter wheat
([micro]g/dry weight g).

Sample                            [H.sub.2]O]

                        Control             Heavy metals


CdS[O.sub.4]      14.36 [+ or -] 0.11   632.79 [+ or -] 0.78
(10 [micro]mol)

ZnS[O.sub.4]      43.53 [+ or -] 0.67   551.93 [+ or -] 0.62
(10 [micro]mol)

CuS[O.sub.4]      25.25 [+ or -] 0.08    68.11 [+ or -] 0.11
(10 [micro]mol)


CdS[O.sub.4]      0.30 [+ or -] 0.01      8.50 [+ or -] 0.08
(10 [micro]mol)

ZnS[O.sub.4]      7.55 [+ or -] 0.08     16.48 [+ or -] 0.20
(10 [micro]mol)

CuS[O.sub.4]      2.24 [+ or -] 0.02      3.22 [+ or -] 0.05
(10 [micro]mol)

Sample                             Stevioside

                        Control             Heavy metals


CdS[O.sub.4]      22.36 [+ or -] 0.12   566.31 [+ or -] 0.81
(10 [micro]mol)

ZnS[O.sub.4]      81.90 [+ or -] 0.54   533.93 [+ or -] 0.63
(10 [micro]mol)

CuS[O.sub.4]      25.22 [+ or -] 0.14    45.71 [+ or -] 0.22
(10 [micro]mol)


CdS[O.sub.4]       0.94 [+ or -] 0.01     7.27 [+ or -] 0.06
(10 [micro]mol)

ZnS[O.sub.4]       8.94 [+ or -] 0.08    11.54 [+ or -] 0.09
(10 [micro]mol)

CuS[O.sub.4]       2.25 [+ or -] 0.04     2.10 [+ or -] 0.04
(10 [micro]mol)

Table 3: Proline content (mg/dry weight g) in roots
of Kazanskaya 560 winter wheat, grown on water
and on stevioside under influence of CdS[O.sub.4],
ZnS[O.sub.4], and CuS[O.sub.4]

Samples                  - Stevioside           + Stevioside

Control              0.004 [+ or -] 0.001   0.016 [+ or -] 0.001
Cu (10 [micro]mol)   0.039 [+ or -] 0.002   0.155 [+ or -] 0.002
Cd (10 [micro]mol)   0.015 [+ or -] 0.001   0.063 [+ or -] 0.001
Zn (10 [micro]mol)   0.009 [+ or -] 0.001   0.041 [+ or -] 0.001
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Title Annotation:Research Article
Author:Nevmerzhitskaya, Julia Yurevna; Mikhailov, Alexander Leonidovich; Strobykina, Anastasia Sergeyevna;
Publication:Biology and Medicine
Article Type:Report
Date:Jul 1, 2014
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