Changes of metal composition of plants treated with brassinosteroids were reported in quite a few experiments. Most of these experiments are related to the ability of brassinosteroids to decrease the intake of heavy metals with plants. 24-epiBL at the concentration of 10-8 mol L-1 in combination with heavy metals blocked metal accumulation in algal cells (Bajguz, 2000) and treatment of Brassica juncea plants with 24- epiBL detoxified the stress generated by NaCl and/or NiCl2 and significantly improved growth, the level of pigments and photosynthetic parameters (Ali et al., 2008b). After foliar application of brassinolide on tomato plants an increase in metals (P, K, Ca and Mg) in aerial parts of plants has been recorded (Nafie & El-Khallal, 2000). Our three-year results showed that after the brassinosteroids treatment of spring wheat some changes of the metals content were determined. However, these changes differed among the experimental years. Brassinosteroids application primarily affected content of K, Mg, Zn and Fe in grain. However, it did not affect Cu content. Brassinosteroids stimulate morphogenesis of plants which causes an increase in leaf area, number of leaves, dry and fresh mass of stems and roots and number of tillers and productive branches. Due to these effects on physiological processes in plants, an increase in the yield and quality of crops production has been observed (Sakurai et al., 1999). Yield increase depends on variety, climatic conditions, soil, application of fertilizers and also on frequency and dates of brassinosteroids application (Khripach et al., 2000, 2003; Janeczko et al., 2010). Different preparations (mixtures of natural 24-epibrassinolide and its synthetic isomers) especially used under unfavourable cultivation conditions cause an increase in yield of crops such as rice, maize, wheat, cotton, tobacco, vegetables and fruit. Exogenous brassinosteroids such as 24-epibrassinolide influences brassinosteroid balance in seedlings of wheat after soaking seeds, drenching or spraying plants and content of endogenous brassinosteroids brassinolide and castasterone varies with leaf insertion and plant age (Janeczko & Swaczynowa, 2010). The relative effects of brassinosteroids may be low, when the conditions under which plants are growing are generally favourable (Khripach et al., 2000). Treatment of barley cultivated in light-textured clay podzolic soil with brassinosteroids in a combination with nitrogen-phosphorus-potassium fertilizer (dose 60 kg N ha-1) increased grain yield by 360 kg ha-1; content of total protein in grain was not affected. However, in our experiments, where NPK fertilization at a dose of 60 kg N ha-1 was applied, no significant increase of grain yield per hectare has been proved. However, the application of brassinosteroids could reduce the negative effect of the stress factors on the yield and dry matter in wheat (Hnilicka et al., 2007, Bajguz, 2009). In a greenhouse experiment with exogenously applied 24-epibrassinolide on two hexaploid wheat (Triticum aestivum L.) cultivars, S-24 (salt tolerant) and MH-97 (moderately salt sensitive), the application of 24-epibrassinolide increased plant biomass and leaf areal per plant of both cultivars under non-saline conditions. However, under saline conditions improvement in growth due to foliar application of 24-epibrassinolide was observed only in salt tolerant cultivar (Shahbaz et al., 2008). Drought stress and high temperature were found to have a negative effect on the amount of dry matter in the above-ground wheat biomass and the yield of grain and straw. Our results regarding the total protein content are in agreement with the results of experiments with wheat after exogenous plant treatment with 24-epibrassinolide, where as well no difference in soluble protein content between control and treated plants after brassinosteroid treatment was determined (Janeczko et al., 2010). In our experiments, no difference was recorded between treated plants and control plants in other qualitative parameters such as gluten content, sedimentation index and bulk density, which are affected more likely by varietals properties, or in Falling number, which is dependent on the harvest date and weather course during the harvest period.

Enhanced resistance of brassinosteroid-treated plants to extreme temperature, salt, pathogens and environmental stresses (heavy metals) was reported by Krishna (2003). The present study revealed the effect of brassinossteroids treatment on the accumulation of Cd, Cu, Pb and Zn contents in aerial wheat biomass or grains. The obtained results are in agreement with the results of Bajguz (2000), who observed that 24-epiBL at the concentration of 10-8 mol L-1 in combination with heavy metals blocked metal accumulation in algal cells. At metal concentrations of 10-6 — 10-4 mol L-1, a combination with 24-epiBL appeared to have a stronger stimulatory effect on a number of cells than a single metal (a stronger inhibitory effect). The inhibitory effect on metal accumulation of 24-epiBL mixed with different heavy metals was arranged in the following order: zinc > cadmium > lead > copper. Our results obtained for spring wheat as an important crop confirm and are complementary to the results of Sharma & Bhardwaj (2007a, b), which describe the effects of 24-epiBL on plant growth, heavy metals uptake in the plants of Brassica juncea L. under heavy metal (Zn, Cu, Mn, Co and Ni) stress. 24-epiBL after the pre-germination treatment blocked copper metal uptake and accumulation in the plants. Likewise results of Anuradha & Rao (2007), obtained in a study on radish (Raphanus sativus L.) after the treatment with 24-epiBL and 28-homobrassinolide clearly indicated the inhibitory influence of brassinosteroids on the cadmium toxicity. Brassinosteroids supplementation alleviated the toxic effect of cadmium and increased the percentage of seed germination and seedling growth. Treatment with brassinosteroids regulates and enhances the activities of antioxidant enzymes ascorbate peroxidase, glutathione reductase, catalase, peroxidase and superoxide dismutase (Sharma, I. et al., 2010) and in drought stressed plants proline and protein content (Behnamnia et al., 2009). The application of brassinosteroids at low concentrations at a certain stage of development reduced significantly the metal absorption in barley, tomatoes and sugar beet. Our results indicate that for the decrease of heavy metals content in plants after the brassinosteroids application the growth stage of spring wheat is very important (Figs. 7 and 8).

The present study shows that the content of heavy metals in wheat plants is reduced variously in different growth stages. The plants of the second group and the third group contained in biomass at the growth stage 73-75 DC lower Pb content as compared to control

plants and the plants of the first group, which was treated with brassinosteroids last at the growth stage 29 — 31 DC. Also in the plants of the second group and the third group at the growth stage 73 — 75 DC lower Cd and Zn contents were determined (with the exception of brassinosteroid 4154 in the third group). The treatment of wheat plants with brassinosteroids 24-epiBL, 24-epiCS and 4154 at the plant growth stage 29-31 DC did not significantly influence content of the heavy metals in aerial plant biomass at the growth stage 47 — 49 DC. In the straw at the growth stage 90-92 DC, lower Pb and Zn contents were subsequently determined only in the plants treated with 24-epiBL and 24-epiCS (Zn also with the application of 4154 in the second group). Lower Cd content was determined only in the variant treated two times with 24-epiBL, which was considered as a highly active brassinosteroid. Lower Pb content was found in the grains of plants of the second group (treated two times in the stages 29-31 DC and 59-60 DC) and the third group (treated once in the stages 59-60 DC).

In terms of the content of heavy metals related to the number and growth stage of brassinosteroids applications, the most effective variants of treatment leading to decrease of

metal content proved either double treatments in the growth stages 29 — 31 DC and 59 — 60 DC (plants of the second group) or one treatment only in the stage 59 — 60 DC (plants of the third group).

Brassinosteroids are able to manage plant water economy during a drought period by decreasing plant activity with a simultaneous conservation of the whole plant for more favourable conditions. Brassinosteroid-treated plants are then able to overcome the drought period in a much better condition than non-treated plants (Sasse, 1999). Their increase in net photosynthetic rate due to brassinosteroids application has already been observed in wheat, tomato and cucumber under normal condition and environment stresses (Ogweno et al., 2008; Shabaz et al., 2008; Xia et al., 2009; Yuan et al., 2010, Hola, 2010). Nowadays biological effects not only naturally occurring brassinosteroids, but also their androstane and pregnane analogues are widely synthesised and their biological effects studied (Hnilickova et al., 2010) as well as their miscellaneous metabolic pathways in plants involving dehydrogenation, demethylation, epimerization, esterification, glycosylation, hydroxylation, side-chain cleavage and sulfonation (Bajguz, 2007). Because brassinosteroids control several
important agronomic traits (Kang & Guo, 2010) such as flowering time, plant architecture, seed yield and stress tolerance, the genetic manipulation of brassinosteroids biosynthesis, conversion or perception offers a unique possibility of both changing plant metabolism and protecting plants from environmental stresses confirming the value of further research on brassinosteroids to improve productivity and quality of agricultural crops (Divi & Krishna,

2009) or their possible use for phytoremediation application (Barbafieri & Tassi, 2010).

2. Conclusion

From the perspective of minimal heavy metals content in biomass and grains related to the number of treatments and growth stage the most effective options of application of brassinolide treatment are those, which lead to a reduction in heavy metals in biomass: either dual treatment in growth stages DC 29-31 and DC 59-60 or single treatment only in the DC 59-60. Favourable is effective reduction of the content of heavy metals in the biomass of plants in grain milk stage (DC 73-75). After treatment of plants with brassinosteroids, when the plants are harvested for ensilage, the content of toxic metals was effectively reduced. Thus, treatment of plants with brassinosteroids can effectively reduce the content of heavy metals in plants (Cd and Pb) or harvested grain (Pb) of wheat and reduce the input of these contaminants into the food chain either cereal or meat products from the food industry. From the point of view of final effect on the content of the heavy metals in plant biomass and grains, the most suitable variant appears to be the single treatment in the growth stage 59-60 DC, which is economically preferable and its final effect does not differ remarkably from double treatments. Likewise lead content in grains decreased in the plants of the second group by 70-74% and of the third group by 48-70%. Thus, treatment of plants with brassinosteroids effectively decreased content of cadmium and lead in wheat plants (biomass) and content of lead in harvested grain and diminished in such way the input of these contaminants into the food chain.

Changes in the minerals content differed according to used brassinosteroid (variant) and investigated year; however unambiguous tendencies of changes or effects were not recorded. In comparison with control plants in the year 2005 the content of minerals in grain of treated plants did not differed significantly. In the year 2006 an increase of K after treatment with 24-epiBL, 4154 and KR1 compounds and a decrease of Zn content after treatment with 24-epiCS and KR1 compounds were recorded. In the year 2007 a decrease of Mg, Mn and Fe content was determined.

Similarly grain quality was not affected by the treatment with brassinosteroids in the investigated years. Content of proteins and gluten in the grains of treated and untreated plants was not significantly different. Similar results were obtained in the sedimentation index and bulk density. Falling number values differed depending on the date of harvest and year of cultivation; in comparison with control plants no difference was recorded. The hypothesis presented is that utilisation of brassinosteroids for plant treatment in the methods of agricultural management with a normal (rational) level of agricultural engineering is not effective. However, by contrast, their application could represent a high economic gain in all cases where the conditions for the cultivation of cereals are not quite ideal, e. g. under conditions of action of different environmental plant stressors, especially with cultivation on soils contaminated with heavy toxic metals or in different arrangements of agricultural engineering. The brassinosteroids-induced enhancement of photosynthetic capacity and regulation of antioxidant enzymes or growth could be under stress factors such saline conditions cultivar specific.