Introduction

Brassinosteroids (BRs) represent a group of naturally occurring steroidal lactones widely distributed in plant kingdom. These compounds, which include the highly bioactive brassinolide (BL) and its derivatives, have been regarded as new plant growth regulators essential for plant development (Mandava 1988). In addition to stimulating growth, BRs have an anti-stress effect on plants. These compounds have wide-range of biological activities that increase the crop yields by changing plant metabolism and protecting plants from environmental stresses (Krishna 2003). BRs have been implicated in protecting plants from various types of stresses like drought (Upreti and Murti 2004), salt (Ozdemir et al. 2004), heat (Dhaubhadel et al. 2002) and heavy metals (Bajguz 2000b; Kaur and Bhardwaj 2004; Janeczko et al. 2005).

Among pollutants of agricultural soils, Cu has become increasingly hazardous due to its involvement in fungicides, fertilizers and pesticides (Chen et al. 2000). However, Cu at high levels become strongly phytotoxic and cause inhibition of plant growth or even death. BRs have ability to regulate the uptake of ions into the plant cells and can be used to reduce the accumulation of heavy metals and radioactive elements. The high biological activity of BRs suggests an important role in the regulation of physiological processes in plants and also in antistress activity. The application of BRs in low concentrations at a certain stage of development reduced the metal absorption in barley, tomatoes and sugerbeet significantly (Volynets et al. 1997). Keeping in mind the anti-stress properties of brassinosteroids, the present piece of work was undertaken to observe the involvement of 24-epibrassinolide in Cu metal stress in B. juncea, an important edible crop.

Materials and methods

Study material and treatments

The seeds of B. juncea L cv. PBR 91 were surface sterilized with 0.01% HgCl2 and rinsed with double distilled water. The sterilized seeds were soaked for 8 h in different concentrations of 24-epiBL (0, 10−7, 10−9 and 10−11 M). In order to study the effects of 24-epiBL on the plant growth and Cu metal uptake in the different parts (leaves, shoots and roots) of the B. juncea plant under Cu stress, a seasonal pot experiment was performed. The garden soil used for investigation was analysed for Cu ion content and filled in the experimental pots (approximately 9 kg in each pot). Different concentrations of copper (CuSO4·5H2O) (0, 25, 50 and 100 mg kg−1 soil) were added to pots containing garden soil. The 24-epibrassinolide treated seeds (8 h) were sown in the pots that contained different concentrations of Cu metal. The plants were kept in natural seasonal conditions. After 30 days of sowing, the plants kept under various treatments were harvested and observations were made for shoot length, fresh and dry weights. Cu-metal uptake was studied in leaves, shoots and roots of the plants.

Copper uptake analysis

Samples of ground, dried plant material were digested using digestion mixture (H2SO4:HNO3:HClO4 in 1:5:1 ratio) by following the method of Allen et al. (1976). The samples were digested till dry colourless residue obtained by evaporating all the acids completely. The dried colourless residue was dissolved in 20 ml double distilled water, which was fine filtered through Whatman # 1 filter paper. The heavy metal analysis was done using Atomic Absorption Spectrophotometer (AA-6200 Shimadzu, Japan). The bioconcentration factor (BCF) was calculated as follows:

$$ {\text{BCF}} = \frac{{{\text{Trace element concentration in plant tissues }}({\text{mg}}\,{\text{kg}}^{{ - 1}} ){\text{ at harvest}}}} {{{\text{Initial concentration of the element in the external nutrient solution (mg}}\,{\text{kg}}^{{{\text{ - 1}}}} )}}. $$

Results were statistically analyzed by calculating the mean value, standard deviation, standard error and student’s t-test following Bailey (1995).

Results

Morphological parameters

The treatment of 24-epiBL to plants under Cu metal stress reduced the toxicity of metal by showing better growth (Table 1). The results indicated that the growth of B. juncea was significantly affected by different concentrations of Cu metal alone against the control. The reduced shoot length was observed in higher treatment of Cu (100 mg kg−1). The pre-germination treatment of seeds with 24-epiBL increased the shoot length. The most effective treatment of 24-epiBL was 10−9 M (Table 1). As the concentration of Cu increased, 10−11 M was found to lower the toxicity of metal by increasing the shoot length of plants. It was revealed that fresh weight of whole plant was increased in all the concentrations of 24-epiBL as compared to control (Table 2). The most effective concentration of 24-epiBL was 10−11 M. Similar observations were made for dry weight of whole plant under the influence of brassinosteroid treatments (Table 2).

Table 1 Effects of 24-epiBL on shoot length (cm) of B. juncea plants under Cu metal stress after 30 days of sowing
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Table 2 Effects of 24-epiBL on fresh and dry weight (g plant− 1) of B. juncea plants under Cu metal stress after 30 days of sowing
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Metal uptake and bioconcentration factor (BCF)

The observations on metal ion content (Cu) in different plant parts (leaves, shoots and roots) revealed that the Cu2+ content of roots was higher, when compared to leaves and shoots in all concentrations of Cu2+ (Fig. 1). Various concentrations (25, 50 and 100 mg kg−1) of Cu2+ in the presence of 24-epiBL treatments, showed a significant reduction in metal uptake and BCF content. In all plant parts (leaves, shoots and roots), uptake of Cu2+ and BCF content was significantly lowered under the effect of different concentrations of 24-epiBL. This compound at 10−7 M concentration was found to be most effective for lowering the Cu uptake and accumulation of ions (Figs. 1, 2). Among all plant parts, the maximum reduction was reported in case of leaves.

Fig. 1
figure 1

Uptake of Cu metal (μg/g DW) in 30-day-old leaves, shoots and roots of B. juncea under the influence of 24-epibrassinolide. Bars represent the SE. Asterisk indicate statistically significant difference from control at P ≤ 0.05

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Fig. 2
figure 2

Bioconcentration factor (BCF) of Cu metal in 30-day-old leaves, shoots and roots of B. juncea under the influence of 24-epibrassinolide. Bars represent the SE. Asterisk indicate statistically significant difference from control at P ≤ 0.05

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Discussion

In the present investigation, 24-epiBL has been observed to improve the growth of B. juncea plant as compared to metal treated and non-treated plants. Similarly, BR stimulated leaf elongation of wheat and mustard plants (Braun and Wild 1984). In addition, Clouse et al. (1992) reported that BR (0.1 μM) induced measurable increase in length of soybean epicotyl. Franck-Duchenne et al. (1998) found that epibrassinolide at a concentration of 0.1 μM promoted stem induction and elongation, and produced large leaves of two cultivars of sweet peppers.

24-Epibrassinolide has also been found to reduce the Cu ion uptake and accumulation in B. juncea L plants. Earlier studies done on the accumulation of heavy metals under the influence of BRs also shown reduction in metal toxicity in barley, tomato and radish etc., (Volynets et al. 1997). Bajguz (2000b) revealed that 24-epibrassinolide blocked the heavy metal accumulation in Chlorella vulgaris cells. It has been observed that brassinosteroids in combination with Pb caused stimulation of phytochelatins synthesis in C. vulgaris. BRs stimulated growth and photosynthetic parameters after blocking the bioabsorption of lead in C. vulgaris (Bajguz 2002). The protective effect of epibrassinolide on winter rape plants under Cd stress was investigated by Janeczko et al. (2005). Bilkisu et al. (2003) reported that BL during Al-related stress stimulated growth in Phaseolus aureus. It was shown that changes in the ions/metal content were influenced by 24-epiBL and dependent on the stage of plant development when the seeds were treated. The content of 137Cs in the plants at the flowering stage was even higher than in the untreated control. Fully matured plants showed some decrease of 137Cs content especially in the vegetative organs probably as a result of vegetative dilution (Khripach et al. 1999).

The mechanism involved for reducing the toxicity may be the chelation of the metal ion by a ligand. Such ligand includes organic acids, amino acids, peptides or polypeptides (Bajguz 2002). Further, the BRs have also influence on electrical properties of membranes and transport of ions by altering their permeability and structure, stability and activity of membrane enzymes. The reduction of toxicity by BRs was associated with lesser uptake of ions and enhanced levels of soluble proteins and nucleic acids with the increasing activity of ATPase, an enzyme responsible for acid secretion and changes in membrane level (Bajguz 2000a). The proton pump generates an H+ electrochemical gradient and provides a driving force for the rapid ion fluxes required for the uptake of various nutrients such as K+, Cl−, NO −3 , amino acids and sucrose across the plasma membrane (Sze et al. 1999). The regulation of H+-ATPase activity (Kasamo 2003) not only allows nutrient uptake in plant cells but also controls water fluxes (Sondergaard et al. 2004). However, little is known about the roles of ion transport systems during BR-induced cell expansion. Previous studies reported a proton secretion induced by BL when applied to azuki bean epicotyls or apical root segments of maize (Zhang et al. 2005). This proton secretion was accompanied by an early hyperpolarization of the plasma membrane, indicating that proton pumps could be targets of BRs (Zhang et al. 2005). The high biological activity of BRs suggests an important role in the regulation of physiological processes in plants and also in anti-stress activity. The present study thus reveals the involvement of 24-epiBL in lowering the uptake of Cu metal ions in B. juncea plants.