Introduction

Cadmium (Cd) is widely used in many fields, including smelting, plating, plastic and dye manufacturing [1–4]. The use has led to the accumulation of Cd in soil [5, 6]. Excess concentration of Cd2+ in soil has caused the decrease in the growth, development and yield of crop [7]. Some toxic symptoms, such as poor growth, stunted growth and chlorosis, have been observed in plants exposed to the excess concentration of Cd2+ [8].

Acid rain has been a well-known environmental problem for decades in the world [9–11]. Acid rain can cause soil acidification [10, 12]. Thus, acid rain directly and indirectly inhibits the growth of crops [9, 13–15]. Many groups have reported the effect of acid rain on the growth of crop and ascertained the maximum pH value of acid rain in which the yield of crop is decreased [9, 13–15]. Acid rain and Cd2+ pollution often occur in the same agricultural region. Meanwhile, acid rain can increase the content of Cd2+ in soil, and the increased Cd2+ can be utilized by crop [16]. Recently, some researchers have studied the combined effect of acid rain and Cd2+ on the growth of crops and have found that acid rain increases the toxic effect of Cd2+ on Vicia faba L. and Phaseolus vulgaris L. [17, 18]. These studies have been conducted using crops at the seedling stage. Seed germination is the beginning of crop life, and it directly affects the growth and biomass of crop [19]. Therefore, it is important to investigate the combined effect of Cd2+ and acid rain on the seed germination of crop.

In the present work, the combined effects of Cd2+ and acid rain on the germination percentage, germination energy, germination index, vigour index, activities of catalase (CAT) and peroxidase (POD), content of malonydiadehyde (MDA) and membrane permeability of soybean during seed germination were investigated. The objective was to reveal the combined effect of Cd2+ and acid rain on the seed germination of soybean. The results can provide theoretical basis for the early monitoring and risk assessment of Cd2+ pollution and acid rain.

Materials and Methods

Plant Material and Treatments

It was reported that the average pH value and concentration of Cd2+ in soil (Jiangsu province, China) was 5.93 and 0.18 mg mg−1, respectively [20]. Thus, six pH values of acid rain (pH 2.5, 3.0, 3.5, 4.0, 5.0, 5.9) and seven concentrations of Cd2+ (0.18, 1.0, 3.0, 6.0, 10.0, 20.0, 50.0 mg L−1) were selected in the present work. Three solutions were prepared as follow: (1) the stock solution of the simulated acid rain at pH 1.0 was prepared with the solution of H2SO4 and HNO3 in the ratio of 4.7 (v/v) by chemical equivalents, according to the general anion composition of rainfall in Southern China [21] and then diluted to pH 2.5, 3.0, 3.5, 4.0, 5.0 and 5.9. (2) The 1 g L−1 cadmium nitrate (Cd(NO3)2) stock solution was freshly prepared by dissolving appropriate quantities of cadmium nitrate tetrahydrate (Cd(NO3)2·4H2O) in deionized water. The solutions of Cd2+ (0.18, 1.0, 3.0, 6.0, 10.0, 20.0, 50.0 mg L−1) were prepared from the stock solution by serial dilution with deionized water. (3) The complex solution of Cd2+ and acid rain was prepared by dissolving appropriate quantities of Cd(NO3)2·4H2O in the simulated acid rain.

Soybean seeds (Su Zao No.1) were obtained from Wuxi Seed CO., LTD, China. Soybean seeds were sterilized with HgCl2 (0.1%) solution for 8 min, rinsed with deionized water for several times. The seeds were divided into four groups and treated with Cd2+ and acid rain: (1) the control group: soybean seeds were soaked in deionized water (pH 7.0); (2) single treatment with acid rain: soybean seeds were soaked in the simulated acid rain at pH 2.5, 3.0, 3.5, 4.0, 5.0 and 5.9; (3) single treatment with Cd2+: soybean seeds were soaked in the solution of Cd2+ (0.18, 1.0, 3.0, 6.0, 10.0, 20.0, 50.0 mg L−1); (4) combined treatment with Cd2+ and acid rain: soybean seeds were soaked in the complex solution of Cd2+ and acid rain. Seeds with the four treatments mentioned above were placed in the culture dishes (pasteurized in oven at 100 °C for 1 h) with filter sheets (θ = 15 cm) to germinate in the culture container kept at a constant temperature of 25 °C. Each dish received 50 healthy seeds. Each treatment was performed in triplicate. During germination, about 30 mL treatment solution (deionized water with pH 7.0, acid rain, Cd2+ solution, the complex solution of Cd2+ and acid rain) was added to each dish, so that about half the volume of each seed was immersed. The treatment solution was replaced every day. Germinated seeds were counted, and germination percentage, germination energy, germination index and vigour index were then calculated after seeds were germinated for a week. Seeds of each treatment were sampled for the determination of the activities of POD and CAT, content of MDA and membrane permeability.

Determination

Germination percentage was: GP = germinated seeds/total seeds × 100.

Germination energy was the germination percentage of seeds on the third day of germination.

Germination index was expressed as Eq. 1:

$$ {\text{GI}} = \sum {\left( {Gt/Dt} \right)} $$
(1)

where GI is the germination index, Gt and Dt are the amount of the germinated seeds and the germination time, respectively.

Vigour index was expressed as Eq. 2:

$$ {\text{VI}} = S \times \sum {\left( {Gt/Dt} \right)} $$
(2)

where VI is the vigour index of seed, S is the growth vigour of seedling in the certain time (expressed with the average fresh weight of a seedling), Gt and Dt are the amount of the germinated seeds and the germination time, respectively.

The level of lipid peroxidation was expressed as the content of MDA [22]. Samples were repeatedly and ultrasonically extracted with ethanol/water (80/20, v/v) containing 1 mg L−1 butylated hydroxytoluene (BHT). After centrifugation, supernatants were pooled, and an aliquot of appropriately diluted sample was added to a test tube with an equal volume of either (1) −TBA solution containing 20% (w/v) trichloroacetic acid (TBA) and 0.01% (w/v) BHT, or (2) +TBA solution containing the above plus 0.65% TBA. Samples were heated at 95°C for 25 min, and after cooling, the absorbance was read at 440, 532 and 600 nm. The content of MDA is expressed as Eq. 3:

$$ {\text{MDA}}\left( {{\text{nmol}}\,{\text{m}}{{\text{l}}^{{ - 1}}}} \right) = {10^6} \times \frac{{A - B}}{{157000}} $$
(3)

where \( \begin{array}{*{20}{c}} {A = \frac{{{\text{Ab}}{{\text{s}}_{{{532} + {\text{TBA}}}}} - {\text{Ab}}{{\text{s}}_{{{600} + {\text{TBA}}}}}}}{{{\text{Ab}}{{\text{s}}_{{{532} - {\text{TBA}}}}} - {\text{Ab}}{{\text{s}}_{{{600} - {\text{TBA}}}}}}}} \hfill \\ {B = \left( {{\text{Ab}}{{\text{s}}_{{{440} + {\text{TBA}}}}} - {\text{Ab}}{{\text{s}}_{{{600} + {\text{TBA}}}}}} \right) \times 0.0571} \hfill \\ \end{array} \)

Here, 157000 was the molar extinction coefficient for MDA. The molar absorbance of 1–10 mM sucrose at 532 and 440 nm was 8.4 and 147, respectively, giving a ratio of 0.0571.

Frozen fresh tissue were ground to fine powder with a mortar and pestle under liquid nitrogen. The proteins were then extracted at 4 °C by grinding with a cold 50 mM potassium phosphate (pH 7.0). The homogenate was centrifuged at 4 °C for 20 min at 12,000×g. The supernatant was used for the measurement of enzymatic activity. The activity of POD was measured by spectrophotometric method [23]. The reaction mixture contained phosphate buffer (pH 7.0, 25 mM), guaiacol (0.05%), 10 mM H2O2, and the activity of POD was determined by the increase in absorbance at 470 nm due to guaiacol oxidation (E = 26.6 mM−1 cm−1). The activity of CAT was measured by titration method [24]. In the measurement, H2O2 was used as reaction substrate. Residual H2O2 was oxidized by KMnO4 (0.1 M) standard solution. The activity of CAT was expressed by the amount of enzyme that decomposes H2O2 per gramme (fresh weight) within 10 min.

Membrane permeability was measured by relative conductivity method described by the previous report [25]. Seeds were rinsed, placed into 40 mL deionized water and gently tumbled at ambient room temperature. Conductance of deionized water was measured after 15 min (C 1 ), 2 h (C 2 ), and 2 h after a freeze–thaw treatment (C total). The rate of electrolyte leakage was expressed as %/h = 100 × (C 2 −C 1 )/(1.75 C total).

Statistical Analysis

Each treatment was performed in triplicate. The data were analyzed through ANOVA using SPSS 11.5 and origin 8.0. Student’s t test was applied to determine the significance between different treatments. Statistical significance was set at the p ≤ 0.05 confidence level.

Results

Germination Indexes of Soybean Seeds

The germination indexes (germination percentage, germination energy, germination index and vigour index) of soybean seeds treated with Cd2+ and acid rain were listed in Table 1. As shown in Table 1, the single treatment with the low concentration of Cd2+ (0.18, 1.0, 3.0 mg L−1) could not change the germination percentage and germination energy of soybean. The single treatment with 6 mg L−1 Cd2+ decreased the germination percentage and germination energy of soybean. The decrease was more obvious with increasing the concentration of Cd2+. The single treatment with acid rain at pH 2.5 severely decreased the germination percentage and germination energy of soybean. The single treatment with acid rain at the other test pH value could not affect the germination percentage and germination energy of soybean. When the soybean seeds were treated with 0.18, 1.0 or 3.0 mg L−1 Cd2+ and acid rain at pH ≤4.0, the germination percentage and germination energy of soybean were decreased compared with those of the control soybean seeds. The results indicated that the combined treatment with the low concentration of Cd2+ and the high pH value of acid rain showed the toxic effect on the seed germination of soybean. The combined treatment with the high concentration of Cd2+ (6.0, 10.0, 20.0, 50.0 mg L−1) and acid rain at all test pH value decreased the germination percentage and germination energy of soybean. The decrease was higher than that of soybean treated with the single Cd2+ or acid rain. The results indicated that the combined treatment with Cd2+ and acid rain increased the potential toxic effect of the single Cd2+ or acid rain on the seed germination of soybean.

Table 1 Germination index of soybean treated with Cd2+ and acid rain
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Germination Index and Vigour Index of Soybean Seeds

It was also observed from Table 1 that the single treatment with the low concentration of Cd2+ (0.18, 1.0, 3.0, 6.0 mg L−1) could not affect the germination index and vigour index of soybean. When the concentration of Cd2+ was increased to 10 mg L−1, the germination index and vigour index of soybean were decreased compared with those of the control soybean. The decrease depended on the increase in the concentration of Cd2+. The change in the germination index and vigour index of soybean treated with the single acid rain was the same as that of the germination percentage and germination energy of soybean. When soybean seeds were treated with Cd2+ and acid rain, germination index and vigour index were decreased compared with those of the control soybean, and the decrease was higher than that of the soybean treated with the single Cd2+ or acid rain.

MDA Content of Soybean Seeds

Figure 1 showed the content of MDA in soybean seeds treated with Cd2+ and acid rain. It was found from Fig. 1 that the treatment with the single Cd2+ firstly decreased and then increased the content of MDA in soybean seeds with increasing the concentration of Cd2+. When the concentration of Cd2+ was 1 mg L−1, the content of MDA in soybean seeds was minimum. When the pH of acid rain was lower than 4.0, the treatment with the single acid rain increased the content of MDA in soybean seeds. When the pH of acid rain was equal to or higher than 4.0, the treatment with the single acid rain could not affect the content of MDA in soybean seeds. When soybean seeds were treated with 1.0 mg L−1 Cd2+ and acid rain at pH 4.0, the content of MDA in soybean seeds was increased compared with that of the control soybean seeds. The combined treatment with 10 mg L−1 Cd2+ and acid rain at all test pH value increased the content of MDA in soybean seeds, and the increase was higher than that of soybean treated with the single Cd2+ or acid rain. The results indicated that the combined treatment with Cd2+ and acid rain increased the potential toxic effect of the single Cd2+ or acid rain on the cell membrane.

Fig. 1
figure 1

Effect of Cd2+ and acid rain on the content of MDA during the seed germination of soybean. ∆ content of MDA shows the difference in the content of MDA between the treatment group and the control group. At the same concentration of Cd2+, values followed by the same lowercase letters are not significantly different at p < 0.05. At the same pH, values followed by the uppercase letters are not significantly different at p < 0.05. 1–7 represent pH 7.0, 5.9, 5.0, 4.0, 3.5, 3.0 and 2.5 acid rain, respectively; (1)–(8) represent 0, 0.18, 1.0, 3.0, 6.0, 10.0, 20.0, 50.0 mg L−1 Cd2+, respectively

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Membrane Permeability of Soybean Seeds

The membrane permeability in soybean seeds treated with Cd2+ and acid rain was shown in Fig. 2. It was observed from Fig. 2 that when the concentration of Cd2+ was higher than 1 mg L−1, the membrane permeability of soybean seeds treated with the single Cd2+ was increased with increasing the concentration of Cd2+. When the concentration of Cd2+ was lower than 10 mg L−1, the membrane permeability was decreased compared with that of the control soybean. The single treatment with acid rain at the high pH value (pH >3.5, except pH 5.9) decreased the membrane permeability of soybean. The opposite phenomenon was observed in soybean treated with the single acid rain at the low pH value (pH ≤3.5). For the combined treatment with Cd2+ and acid rain, the membrane permeability in soybean was higher than that of the control soybean, and the increase was higher than that of the single treatment with Cd2+ or acid rain. The results indicated that acid rain aggravated the damage to the cell membrane of soybean exposed to Cd2+; in turn, Cd2+ also aggravated the damage to the cell membrane of soybean under acid rain.

Fig. 2
figure 2

Effect of Cd2+ and acid rain on the membrane permeability during the seed germination of soybean. ∆ membrane permeability shows the difference in the membrane permeability between the treatment group and the control group. At the same concentration of Cd2+, values followed by the same lowercase letters are not significantly different at p < 0.05. At the same pH, values followed by the uppercase letters are not significantly different at p < 0.05. 1–7 represent pH 7.0, 5.9, 5.0, 4.0, 3.5, 3.0 and 2.5 acid rain, respectively; (1)–(8) represent 0, 0.18, 1.0, 3.0, 6.0, 10.0, 20.0, 50.0 mg L−1 Cd2+, respectively

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Antioxidant Enzyme Activities of Soybean Seeds

The activities of CAT and POD in soybean treated with Cd2+ and acid rain were given in Figs. 3 and 4, respectively. It was shown in Figs. 3 and 4 that the single treatment with the low concentration of Cd2+ (0.18, 1, 3, 6 mg L−1) increased the activities of CAT and POD in soybean during seed germination, indicating that the single treatment with the low concentration of Cd2+ increased the ability of CAT and POD to remove free radicals. The opposite effect was observed in the activities of CAT and POD in the single treatment with the high concentration of Cd2+ (≥10 mg L−1), indicating that the single treatment with the high concentration of Cd2+ decreased the ability of CAT and POD to remove the free radicals. It was also observed from Figs. 3 and 4 that compared with those of the control soybean, the activities of CAT and POD in soybean treated with the single acid rain at pH 4.0–5.9 were increased, while the activities of CAT and POD in soybean treated with the single acid rain at pH 2.5, 3.0 and 3.5 were decreased. For the combined treatment with Cd2+ and acid rain, the minimum concentration of Cd2+ in which the activities of CAT and POD were obviously lower than that of the control soybean was smaller than that of soybean treated with the single Cd2+. Meanwhile, the pH of acid rain at which the activities of CAT and POD was obviously lower than that of the control soybean was higher than that of soybean treated with the single acid rain. Obviously, the combined effect of Cd2+ and acid rain on the activities of CAT and POD was more obvious than that of the single Cd2+ or acid rain.

Fig. 3
figure 3

Effect of Cd2+ and acid rain on the activity of CAT during the seed germination of soybean. ∆ activity of CAT shows the difference in the activity of CAT between the treatment group and the control group. At the same concentration of Cd2+, values followed by the same lowercase letters are not significantly different at p < 0.05. At the same pH, values followed by the uppercase letters are not significantly different at p < 0.05. 1–7 represent pH 7.0, 5.9, 5.0, 4.0, 3.5, 3.0 and 2.5 acid rain, respectively; (1)–(8) represent 0, 0.18, 1.0, 3.0, 6.0, 10.0, 20.0, 50.0 mg L−1 Cd2+, respectively

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Fig. 4
figure 4

Effect of Cd2+ and acid rain on the activity of POD during activities of the seed germination of soybean. ∆ activity of POD shows the difference in the activity of POD between the treatment group and the control group. At the same concentration of Cd2+, values followed by the same lowercase letters are not significantly different at p < 0.05. At the same pH, values followed by the uppercase letters are not significantly different at p < 0.05. 1–7 represent pH 7.0, 5.9, 5.0, 4.0, 3.5, 3.0 and 2.5 acid rain, respectively; (1)–(8) represent 0, 0.18, 1.0, 3.0, 6.0, 10.0, 20.0, 50.0 mg L−1 Cd2+, respectively

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Discussions

It is well known that germination percentage is an important indicator used to evaluate seed quality. Germination energy represents the seed vigour. Germination index and vigour index can reflect the germination status. Here, germination percentage, germination energy, germination index and germination vigour were selected to evaluate the combined effect of Cd2+ and acid rain on the seed germination of soybean. It was found that acid rain at pH ≥3.0 or the low concentration of Cd2+ (0.18 and 1 mg L−1) could not change the germination percentage, germination energy, germination index and vigour index of soybean seeds; thus, these treatments could not affect the seed germination of soybean (Table 1). These concentrations of Cd2+ and the pH values of acid rain were safe to the seed germination of soybean. But the combined treatment with acid rain at pH 3.5, 4.0 and the low concentration of Cd2+ (0.18 and 1 mg L−1) decreased the germination percentage, germination energy, germination index and vigour index of soybean (Table 1). The results indicated that the combined treatment with the safe concentration of Cd2+ and acid rain (H+) has potential threat to the seed germination of soybean. Moreover, the combined treatment with the safe pH value of acid rain (such as pH 5.0 or 4.0) and the high concentration of Cd2+ (≥3 mg L−1) decreased the seed germination of soybean. The same effect was observed in the combined treatment with the safe concentration of Cd2+ (0.18, 1 mg L−1) and the low pH value of acid rain (pH 3.0, 2.5). The results indicated that when the soil is with the safe pH value and the high concentration of Cd2+, the soil could threaten the seed germination of soybean. When the concentration of Cd2+ in the soil is safe and the pH value in the soil is low, the soil could also threaten the seed germination of soybean. Furthermore, the combined treatment with the high concentration of Cd2+ (≥3 mg L−1) and the low pH value of acid rain (pH 3.0, 2.5) decreased the seed germination of soybean, and the decrease was more obvious than that of the single treatment with the high concentration of Cd2+ or the low pH value of acid rain. These results indicated that the combined pollution of Cd2+ (≥3 mg L−1) and acid rain (pH 3.0, 2.5) has more threat to the seed germination of soybean than that of the single pollution of Cd2+ or acid rain.

The cell membrane is a selective permeable boundary around a cell or an organelle [26]. It controls the substances in and out cell and organelle, transfers signals from the outside to inside of cell, participates in the synthesis and assembly of substance in the cell and provides physical connections for cell construction and substances in the outside of cells[26]. Thus, the seed germination of plant depends on the integrity of structure and stability of function in the cell membrane. It has been reported that the abiotic stress can cause the excess accumulation of free radicals in plants [27, 28]. The excess free radicals oxidize the unsaturated fatty acids in the membrane lipid of cell [29], leading to the peroxidation of cell membrane lipid [30, 31]. Subsequently, the cell membrane is damaged, the selectivity of the cell membrane is destroyed and then the electrolyte leakage from the cytoplasm (the membrane permeability) is increased. Hence, the content of MDA and membrane permeability can reflect the injury degree of the cell membrane [30, 31]. CAT and POD are two major antioxidant enzymes, and they are relative to the seed germination of plant [19]. CAT and POD can effectively remove the free radicals (including O2−, OH−, H2O2, etc.) induced by the abiotic stress in order to protect the plant cell from the damage of abiotic stress [28]. It was found from our experimental results that the treatment with the single acid rain (pH ≥3.5) or Cd2+ (<6 mg L−1) increased the activity of CAT and POD in soybean (Figs. 3 and 4). The increase could lead to the increase in the ability of CAT and POD to remove free radicals and then could make the soybean seeds maintain a balance in the production and scavenging of free radicals. Consequently, the content of MDA and cell membrane permeability was not obviously changed (Figs. 1 and 2), indicating the cell membrane was not damaged. Thus, the single treatment with the low concentration of Cd2+ and the high pH value of acid rain could not affect the seed germination of soybean (Table 1). The decrease in the activities of CAT and POD was observed in the single treatment with the low pH value of acid rain (such as pH 2.5) and the high concentration of Cd2+ (≥10 mg L−1). The decrease in the activities of CAT and POD led to the increase in the content of MDA and membrane permeability and then the decrease in the seed germination of soybean. The combined treatment with Cd2+ and acid rain (including the safe concentration of Cd2+ and the safe pH value of acid rain) decreased the activities of CAT and POD, and then increased the content of MDA and the cell membrane permeability. The toxic effect was more obvious than that of the treatment with the single Cd2+ or acid rain. Thus, the decrease in the seed germination of soybean in the combined treatment with Cd2+ pollution and acid rain was resulted in the oxidative stress induced by the decrease in the activities of CAT and POD of soybean.