Effects of bisphenol A on growth and nitrogen nutrition of roots of soybean seedlings

Authors

  • Hai Sun,

    1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China
    2. School of Environmental and Civil Engineering, Jiangnan University, Wuxi, China
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  • Lihong Wang,

    1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China
    2. School of Environmental and Civil Engineering, Jiangnan University, Wuxi, China
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  • Qing Zhou

    1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China
    2. School of Environmental and Civil Engineering, Jiangnan University, Wuxi, China
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Abstract

Bisphenol A (BPA) is an environmental endocrine disruptor that seriously threatens ecological systems. Plants are the primary producers in ecological systems, but little information is available concerning the toxic effect of BPA on plants. In the present study, the effects of BPA on the growth and nitrogen nutrition of roots of soybean seedlings were investigated by using a root automatic scan apparatus and biochemical methods. It was found that when soybean seedlings were treated with 1.5 mg/L BPA, the growth of roots was improved, the content of nitrate in roots was increased, the content of ammonium in roots was decreased, and the activities of nitrate reductase and nitrite reductase in roots were not changed. The opposite effects were observed in roots treated with 17.2 mg/L and 50.0 mg/L BPA, except for an increase in the content of nitrate in roots treated with 17.2 mg/L BPA and a decrease in the activities of nitrate reductase and nitrite reductase in roots of soybeans seedlings. Statistical analysis indicated that the change in the nitrogen nutrition of roots of soybean seedlings treated with BPA was one reason why the growth of roots was changed. The authors suggest that the potential environmental and ecological risk of BPA to plants should receive more consideration. Environ. Toxicol. Chem. 2013;32:174–180. © 2012 SETAC

INTRODUCTION

Endocrine-disrupting chemicals have attracted considerable attention because they are widely distributed and easily accumulated in ecosystems and exert adverse effects on ecosystems 1–3. As a representative endocrine-disrupting chemical, bisphenol A (BPA; 4,4′-isopropylidine diphenol) is an intermediate in the industrial production of resins, plastics, and coatings 4. The global production capacity of BPA is approximately 3,700,000 tons per year 5, and it is predicted that there is a 6 to 10% yearly growth in the demand for BPA 6. Because of its large-scale production and extensive application, BPA has been found ubiquitously in the environment 7. Many ecosystems, including surface, river, waste, and drinking waters, have been polluted with BPA 1, 8, 9. It has been reported that the concentration of BPA in hazardous waste landfill leachates is 17.2 mg/L 10. Because sewage water and sludge are increasingly used for the irrigation and amendment of soil, BPA has also been accumulated in soil 4. Although the accumulated BPA is required by national regulations to be captured and treated in some regions, unexpected incidents also can happen in BPA factories in these regions. The accumulation of BPA in ecosystems and the incidents of pollution with BPA seriously threaten the environment and human health 11.

The effects of BPA on animals and humans have been well documented. It has been reported that the morphology, habitual behavior, and histological structure of salmon yolk-sac fry exposed to BPA at three concentrations (10, 100, and 1,000 µg/L) have been obviously changed, and edema and hemorrhage of the yolk sac have been observed 12. Crain et al. 7 reported that BPA at the environmentally relevant concentration of 21 µg/L or less can disrupt the endocrine system of many living organisms. Suzuki and Hattori 13 demonstrated that BPA can cause a decrease in the calcium level and calcitonin secretion in plasma of goldfish, suppressing the activities of tartrate-resistant acid phosphatase and alkaline phosphatase b. Kurebayashi et al. 14 found trace disposition of BPA in adult, pregnant, and neonatal rats. Domoradzki et al. 15 reported the pharmacokinetics and metabolism of BPA at three different stages of pregnancy in Sprague–Dawley rats. Other concerns regarding human exposures to BPA come from reports on the measurement of the aglycone form of BPA in maternal and fetal tissues 16, 17. In addition, it has been reported that tobacco seedlings can absorb BPA through root systems and metabolize BPA to the beta-glucoside of BPA 18. Although the metabolic mechanism of BPA in plants remains unknown, the metabolic products of BPA can be considered as detoxified forms 19. From the studies mentioned above, we can conclude that BPA can also affect plants, but few reports on the potential toxic effects of BPA on plants have been presented 18, 19 compared with the number of studies on the toxicity of BPA on animals 20, 21. As we know, plants are the primary producers in ecosystems, and they synthesize organic substances and provide the energy for ecosystems. The roots of plants are a vital organ formed by long-term adaptation to land conditions. They can anchor the plant and absorb water and nutrients. While doing so, the roots directly contact BPA. Thus, it is important to investigate the effect of BPA on plant roots.

Nitrogen is not only a plant nutritional substance but also the basis of the growth and development of plants. In the present study, the effects of BPA on the growth of roots and the nitrogen nutrition in roots were investigated; soybean (Glycine max), an important economic crop, was selected as a model plant. Our results provide some guidelines for elucidating the effect mechanism of BPA on the growth of plants and also provide a theoretical and experimental foundation for scientifically evaluating the ecological risk in farmland where BPA occurs frequently. The results also have prospective significance for foreseeing the environmental hazards of the accumulation of BPA in soil, particularly for the evaluation of environmental hazards caused by acute BPA pollution.

MATERIALS AND METHODS

Preparation of BPA solution

In our pre-experiment (results not shown), we used a BPA solution at concentrations of 1.5, 7.0, 12.0, 17.2, and 50.0 mg/L to treat soybean seedlings. The growth of roots was improved in soybean seedlings treated with 1.5 mg/L BPA, and the growth of roots was inhibited in soybean seedlings treated with 7.0, 12.0, 17.2, or 50.0 mg/L BPA. In the present study, to elucidate the effect mechanism of BPA on plants, the following three representative concentrations of BPA (1.5, 17.2, 50.0 mg/L) were selected: 1.5 mg/L is the safe concentration for drinking water, calculated according to the upper limit of safety for humans reported by the U.S. Environmental Protection Agency (U.S. EPA) 22; 17.2 mg/L is the concentration in hazardous landfill leachates 10; and 50.0 mg/L is a possible concentration of BPA accumulated in soil in the future 4 or the concentration of BPA in soil suddenly polluted by BPA at high concentration, as well as the high concentration of BPA selected in other research on the toxic effect of BPA on plants 4. At 25°C, BPA is a solid compound with low volatility and water solubility ranging from 120 to 300 mg/L 4, 5. Different concentrations of BPA solutions (1.5, 17.2, and 50.0 mg/L) were prepared by dissolving appropriate quantities of BPA (Sinopharm Chemical Reagent) in Hoagland's solution (pH 7.0) with continuous stirring and/or sonication at 25°C. All reagents used were of analytical grade.

Plant culture and treatments

Seeds of soybean (Zhonghuang 25; Wuxi Seed) were sterilized in HgCl2 (0.1%) solution for 5 min and rinsed with distilled water several times. Then, the seeds were placed in a dish atop three layers of gauze and germinated in the incubator at 25 ± 1°C. When the length of the radicle was approximately 1 cm, seedlings were transplanted into plastic pots (diameter 15 cm, three plants per pot) filled with distilled water. The distilled water was renewed every day. After the second true leaf had developed (approximately 10 d after germination), the seedlings were cultured in one-half strength Hoagland's solution (pH 7.0) in the greenhouse. The photosynthetic photon flux density provided by the incandescent lamps at the greenhouse was 300 µmol/m2/s as measured with a photometer (Fluke 941). The nutrient solution was aerated twice per day with an electronic air pump, and distilled water was added to maintain the solution volume 23. The nutrient solution was renewed every 3 d to stabilize pH value. After the third true leaf had developed (approximately 30 d after germination), the soybean seedlings were transplanted to BPA solution at the different concentrations (1.5, 17.2, and 50.0 mg/L, pH 7.0). The control soybean seedlings were cultured in one-half strength Hoagland's solution (pH 7.0) without BPA. All treatments were performed in triplicate. The solution was renewed every 3 d. Soybean seedlings were cultured in the BPA solution for 7 d and then cultured in one-half strength Hoagland's solution (pH 7.0) without BPA, to be restored from day 8 to day 14. After treatment for 7 and 14 d, the roots of soybean seedlings were collected for determination of the length, surface area, volume, fresh and dry weights, content of nitrate, content of ammonium, activity of nitrate reductase (NR), and activity of nitrite reductase (NiR).

Determination of the growth indices in soybean seedlings

The growth indices include the length, surface area, volume, and fresh and dry weights of roots for each plant. Root morphological parameters were determined by using a root automatic scan apparatus (Perfection V700 Photo; Seiko Epson) equipped with WinRHIZO software (Ver 2009a; Regent Instruments). WinRHIZO2009a is software that recognizes digital root images and analyzes root parameters (length, surface area, and volume). For each replication, roots of nine plants were analyzed 24. Dry weights of roots were determined after drying in an oven for 12 h at 80°C.

Measurement of the content of nitrate

Root samples (1 g), together with 50 mg polyvinylpolypyrrolidone were homogenized with 1 ml saturated borax and 5 ml double-distilled water and then heated for 15 min in a boiling water bath. After the root samples cooled, 2 ml potassium ferrocyanide (0.25 M) and 2 ml zinc acetate (1 M) were added. The mixture was then centrifuged at room temperature for 10 min (10,000 g). The content of nitrate was measured using reduction of nitrate by vanadium (III) combined with detection by the acidic Griess reaction 25. The content of nitrate was expressed as micrograms per gram fresh weight.

Measurement of the content of ammonium

The content of ammonium (NHmath image) was determined spectrophotometrically by using Nessler's reagent method 26. Root samples (500 mg) from the control and treated seedlings were homogenized in 0.3 mM H2SO4 and centrifuged (20,000 g) for 20 min. The supernatant was used for NHmath image estimation. The reaction mixture (2.7 ml) contained 0.1 ml extract, 0.1 ml 10% (w/v) potassium sodium tartrate, 2.4 ml double-distilled water, and 0.1 ml Nessler's reagent. After 5 min of incubation, absorbance of reaction mixtures was recorded at 425 nm. The NHmath image content was calculated using a standard curve prepared with NH4Cl.

Assay of the activities of NR and NiR

The roots of soybean seedlings were homogenized in three volumes of extraction buffer according to the method of Ida et al. 27. The extraction buffer contained 50 mM Tris-HCl (pH 7.9), 5 mM cysteine, and 2 mM ethylenediaminetetraacetic acid (EDTA). The homogenate was centrifuged at 10,000 g for 20 min, and the supernatant (500 µl) was concentrated with a Microcon 10 (Amicon) to reduce the number of nitrate ions. The concentrated supernatant was diluted by the addition of 500 µl buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM cysteine, and 2 mM EDTA, and was assayed for NR and NiR activity. The procedures described above were carried out at 4°C. The enzyme activity was assayed by an in vitro method. The assay mixture for NR contained 25 mM potassium phosphate buffer (pH 7.5), 10 mM KNO3, 0.2 mM reduced nicotine adenine dinucleotide (NADH), 5 mM NaHCO3, and 5 µl of extract in a final volume of 0.5 ml. The assays were conducted at 30°C for 15 min. The reaction was terminated by the addition of 50 µl of 0.5 M Zn(CH3COO)2, and the excess NADH was oxidized by the addition of 50 µl of 0.15 mM phenazine methosulfate 28. The mixture was centrifuged at 10,000 g for 5 min, and the amount of NOmath image produced was measured by combining 500 µl of the supernatant with 250 µl of 1% sulfanylamide prepared in 1.5 N HCl and 250 µl of 0.02% N-(1-naphthyl)ethylene-diamine dihydrochloride and reading at 540 nm in a spectrophotometer 29.

The activity of NiR was assayed by following the reduction of NOmath image in the assay mixture 30. The assay mixture contained 50 mM Tris-HCl (pH 7.5), 0.5 mM NaNO2, 1 mM methyl viologen, and 50 µl extract in a final volume of 0.9 ml. The reaction was started by the addition of 100 µl of 0.12 M Na2S2O4 dissolved in 0.2 M NaHCO3 and incubated at 30°C for 60 min. The reaction was terminated by vigorous vortexing until the color of the methyl viologen had disappeared completely. After the addition of 100 µl of 1 M Zn(CH3COO)2, the mixture was centrifuged at 10,000 g for 10 min. The residual NOmath image in the supernatant was determined as in the assay of the NR activity.

Statistical analysis

Differences between the treatments were analyzed by one-way analysis of variance (ANOVA) in SPSS 16.0 and Origin 8.0. Student's t test was applied to determine the significance between different treatments (p ≤ 0.05).

RESULTS

Effects of BPA on the growth of roots of soybean seedlings

Table 1 shows the results of the growth indices in roots of soybean seedlings treated with BPA at the different concentrations on the seventh day. When soybean seedlings were treated with 1.5 mg/L BPA, the root length, root surface area, root volume, and fresh and dry weights of roots were increased by 10.10, 5.47, 8.66, 0.78, and 4.35%, respectively, compared with the control. When soybean seedlings were treated with 17.2 mg/L BPA, the root length, root surface area, root volume, and fresh and dry weights of roots were decreased by 28.59, 31.74, 28.14, 22.27, and 17.39%, respectively, compared with the control. When soybean seedlings were treated with 50.0 mg/L BPA, the indices mentioned above were decreased by 68.04, 57.96, 61.90, 47.27, and 47.83%, respectively, compared with the control.

Table 1. Effects of bisphenol A (BPA) on the five growth indices in soybean seedlings on day 7a
BPA (mg/L)Root length (cm)Root surface area (cm2)Root volume (cm3)Fresh weights of roots (g)Dry weights of roots (g)
  • a

    Values are means ± standard deviations, n = 9. Differences at p < 0.05 are shown with different letters on the same line.

01,001.82 ± 3.42A (100.00)199.47 ± 0.66A (100.00)2.31 ± 0.01A (100.00)2.56 ± 0.06A (100.00)0.23 ± 0.01A (100.00)
1.51,102.96 ± 4.91B (110.10)210.38 ± 3.21B (105.47)2.51 ± 0.01D (108.66)2.58 ± 0.10A (100.78)0.24 ± 0.01A (104.35)
17.2715.36 ± 2.90C (71.41)136.15 ± 1.85C (68.26)1.66 ± 0.01C (71.86)1.99 ± 0.05B (77.73)0.19 ± 0.01B (82.61)
50.0320.18 ± 0.89D (31.96)83.86 ± 0.40D (42.04)0.88 ± 0.01D (38.10)1.35 ± 0.07C (52.73)0.12 ± 0.02C (52.17)

On day 14, the effect of BPA on root growth was observed (Table 2). When soybean seedlings were treated with 1.5 mg/L BPA, the root length, root surface area, root volume, and fresh and dry weights of roots were increased by 9.47, 6.13, 7.62, 1.92, and 9.09%, respectively, compared with the control. The root length, root surface area, root volume, and fresh and dry weights of roots of soybean seedlings treated with 17.2 mg/L BPA were decreased by 22.26, 24.91, 23.31, 9.96, and 9.09%, respectively, compared with the control. When soybean seedlings were treated with 50.0 mg/L BPA, the indices mentioned above were decreased by 65.12, 57.09, 62.29, 41.76, and 45.45%, respectively, compared with the control. Given these results and the results from the stress period (Table 1), we conclude that the decreases in the root length, root surface area, root volume, and fresh and dry weights of roots were slightly alleviated during the recovery period.

Table 2. Effects of bisphenol A (BPA) on the five growth indices in soybean seedlings on day 14a
BPA (mg/L)Root length (cm)Root surface area (cm2)Root volume (cm3)Fresh weights of roots (g)Dry weights of roots (g)
  • a

    Values are means ± standard deviations, n = 9. Differences at p < 0.05 are shown with different letters on the same line.

01,033.24 ± 1.04A (100.00)202.18 ± 0.95A (100.00)2.36 ± 0.01A (100.00)2.61 ± 0.03A (100.00)0.22 ± 0.01AB (100.00)
1.51,131.08 ± 1.55B (109.47)214.57 ± 1.25B (106.13)2.54 ± 0.02B (107.63)2.66 ± 0.04A (101.92)0.24 ± 0.01A (109.09)
17.2803.26 ± 3.05C (77.74)151.82 ± 1.97C (75.09)1.81 ± 0.01C (76.69)2.35 ± 0.01B (90.04)0.20 ± 0.01B (90.91)
50.0360.35 ± 0.89D (34.88)86.76 ± 0.77D (42.91)0.89 ± 0.01D (37.71)1.52 ± 0.02C (58.24)0.12 ± 0.01C (54.55)

Effects of BPA on the content of nitrate in roots of soybean seedlings

Figure 1 shows the results for the content of nitrate in roots of soybean seedlings treated with BPA. The content of nitrate in roots treated with 1.5 mg/L BPA was increased by 22.89% compared with the control. When soybean seedlings were treated with 17.2 mg/L BPA, the content of nitrate was significantly increased by 104.83% compared with the control. Treatment with 50.0 mg/L BPA led to a decrease in the content of nitrate by 60.30%.

Figure 1.

Effects of bisphenol A (BPA) on the content of nitrate in roots of soybean seedlings. Results are means ± standard deviations (n = 3). Differences at p < 0.05 are shown with different letters.

After 7 d of recovery, the content of nitrate in roots of soybean seedlings treated with 1.5 mg/L BPA was increased by 35.54% compared with the control. Obviously, the increase was greater than that in the stress period. The increase in the content of nitrate in roots of soybean seedlings treated with 17.2 mg/L BPA (119.64%) after 7 d of recovery was greater than that in the stress period (104.83%). The content of nitrate in roots of soybean seedlings treated with 50.0 mg/L BPA was decreased by 79.48% compared with the control, which was greater than the decrease in the stress period.

Effects of BPA on the content of ammonium in roots of soybean seedlings

The contents of ammonium in roots of soybean seedlings treated with BPA at the different concentrations on day 7 are shown in Figure 2. The content of ammonium in roots of soybean seedlings treated with 1.5 mg/L BPA was decreased by 7.37% compared with the control. Increases of 7.55 and 26.26% in the content of ammonium in roots of soybean seedlings treated with 17.2 mg/L and 50.0 mg/L BPA, respectively, were observed compared with the control.

Figure 2.

Effects of bisphenol A (BPA) on the content of ammonium in roots of soybean seedlings. Results are means ± standard deviations (n = 3). Differences at p < 0.05 are shown with different letters.

The contents of ammonium in roots of soybean seedlings treated with BPA at the different concentrations after 7 d of recovery are also shown in Figure 2. We observed that after the 7-d recovery, the content of ammonium in roots of soybean seedlings treated with 1.5 mg/L BPA was decreased by 7.72% compared with the control. The contents of ammonium in roots of soybean seedlings treated with17.2 mg/L and 50.0 mg/L BPA were increased by 19.84 and 12.13%, respectively, compared with the control.

To understand the relationship between the growth and nitrogen nutrition in soybean seedlings treated with BPA, the correlation coefficients between the growth indices and the content of ammonium in the stress and recovery periods were calculated. For the stress period, the linear regression equation and correlation coefficient (r) of the root length (root surface area, root volume, and fresh and dry wts of roots) and the content of ammonium were y = −57.892x + 3,341.308 and r = 0.990 (y = −9.541x + 578.751, r = 0.970; y = −0.121x + 7.179, r = 0.985; y = −0.194x + 9.926, r = 0.832; y = −0.009x + 0.597, r = 0.960, respectively; Table 3). For the recovery period, the linear regression equation and correlation coefficient of the root length (root surface area, root volume, fresh and dry wts of roots) and the content of ammonium were y = 44.454x − 873.492 and r = 0.462 (y = 8.708x − 170.243, r = 0.534; y = 0.102x − 1.996, r = 0.489; y = 0.057x + 0.091, r = 0.384; y = 0.006x − 0.017, r = 0.386, respectively; Table 3). The results indicate that in the stress period, the five growth indices were negatively correlated with the content of ammonium, and the relationship reached a significant level (p < 0.01).

Table 3. Relationship between the growth indices and ammonium content of soybean seedlings treated with bisphenol A in the stress period (7 d) and the recovery period (14 d) a
Stress period (7 d)Recovery period (14 d)
Linear regression equationCorrelation coefficient (r)Linear regression equationCorrelation coefficient (r)
  • a

    In the table, x represents the content of ammonium. y1, y2, y3, y4, and y5 represent root length, root surface area, root volume, fresh dry weights of root, and dry weights of root, respectively.

y1 = −57.892x + 3,341.3080.990y1 = 44.454x − 873.4920.462
y2 = −9.541x + 578.7510.970y2 = 8.708x − 170.2430.534
y3 = −0.121x + 7.1790.985y3 = 0.102x − 1.9960.489
y4 = −0.194x + 9.9260.832y4 = 0.057x + 0.0910.384
y5 = −0.009x + 0.5970.960y5 = 0.006x − 0.0170.386

Effects of BPA on the activity of NR in roots of soybean seedlings

After 7 d of stress with BPA, the activity of NR in roots of soybean seedlings treated with 1.5 mg/L BPA was not obviously changed in comparison with the control (Fig. 3). The activities of NR in roots of soybean seedlings treated with 17.2 mg/L and 50.0 mg/L BPA were decreased by 64.93 and 89.83% compared with the control, respectively (Fig. 3).

Figure 3.

Effects of bisphenol A (BPA) on the activity of nitrate reductase (NR) in roots of soybean seedlings. Results are means ± standard deviations (n = 3). Differences at p < 0.05 are shown with different letters.

After 7 d of recovery, the activity of NR in roots of soybean seedlings treated with 1.5 mg/L BPA was restored to the normal level (Fig. 3). The activities of NR in roots of soybean seedlings treated with 17.2 mg/L and 50.0 mg/L BPA were decreased by 35.89 and 93.53% compared with the control, respectively (Fig. 3). The decrease in the activity of NR in roots of soybean seedlings treated with 17.2 mg/L BPA was alleviated, whereas the decrease in the activity of NR in roots of soybean seedlings treated with 50.0 mg/L BPA could not be alleviated (Fig. 3).

To understand the relationship between the content of ammonium and the activity of NR in roots of soybean seedlings treated with BPA, the correlation coefficients between the content of ammonium and the activity of NR in the stress and recovery periods were calculated. For the stress period, the linear regression equation and correlation coefficient of the content of ammonium and the activity of NR were y = −0.372x − 52.436 and r = 0.949. For the recovery period, the linear regression equation and correlation coefficient of the content of ammonium and the activity of NR were y = 0.116x + 35.601 and r = 0.507. The results indicated that in the stress period, the content of ammonium was negatively related to the activity of NR, and the relationship reached significance (p < 0.01).

Effects of BPA on the activity of NiR in roots of soybean seedlings

After 7 d of stress, the activity of NiR in roots of soybean seedlings treated with 1.5 mg/L BPA was not obviously changed in comparison with the control. When soybean seedlings were treated with 17.2 mg/L and 50.0 mg/L BPA, the activities of NiR in roots were decreased by 15.29 and 29.90% compared with the control, respectively (Fig. 4).

Figure 4.

Effects of bisphenol A (BPA) on the activity of nitrite reductase (NiR) in roots of soybean seedlings. Results are means ± standard deviations (n = 3). Differences at p < 0.05 are shown with different letters.

After 7 d of recovery, the activities of NiR in soybean seedlings treated with 1.5, 17.2, and 50.0 mg/L BPA were decreased by 1.13 (which did not reach significance), 22.37, and 33.76%, respectively, compared with the control. Obviously, after 7 d of recovery, the decreases in the activities of NR in roots of soybean seedlings treated with 17.2 mg/L and 50.0 mg/L BPA in the stress period were not alleviated.

To understand the relationship between the content of ammonium and the activity of NiR in soybean seedlings treated with BPA, the correlation coefficients between the content of ammonium and the activity of NiR in the stress period and recovery period were calculated. For the stress period, the linear regression equation and correlation coefficient of the content of ammonium and the activity of NiR were y = −0.104x + 79.384 and r = 0.970. For the recovery period, the linear regression equation and correlation coefficient of the content of ammonium and the activity of NiR were y = 0.039x + 25.339 and r = 0.706. The results indicated that in the stress period, the content of ammonium was negatively related to the activity of NiR, and the relationship reached a significant level (p < 0.01).

DISCUSSION

The root length, root surface area, root volume, and fresh and dry weights of root can directly reflect the growth status of roots. Our results show the effect of BPA on the root growth of soybean seedlings. When soybean seedlings were treated with BPA at a low concentration (1.5 mg/L), the growth of the roots of soybean seedlings was improved; however, when soybean seedlings were treated with BPA at high concentrations (17.2 and 50.0 mg/L), the growth of the roots was inhibited. The findings were in agreement with previous reports on durum wheat and lettuce 4. We suggest that, in evaluating the ecological risk of BPA pollution, the toxic effect of BPA on plants should be considered more carefully.

It is well known that the growth of plant roots results from a combination of three processes at the cellular level: cell expansion, division, and differentiation. The three processes were regulated by the level of proteins in vivo 31. Nitrogen is a very important inorganic nutrient in plants and a major constituent of amino acids and proteins 32. Plants easily take up NOmath image through their roots and integrate it into the various plant proteins. Before integration into plant proteins, nitrate is reduced to ammonium in a two-step process catalyzed by NR and NiR 33, 34. In the present work, the contents of nitrate and ammonium, as well as the activities of NR and NiR, were determined to clarify the effect mechanism of BPA on the growth of roots of soybean seedlings. Our correlation analysis results indicate that the growth of roots of soybean seedlings treated with BPA was related to the content of ammonium, and the content of ammonium was regulated by the activities of NR and NiR. The effect mechanism depended on the concentration of BPA. The 1.5 mg/L BPA induced an increase in the content of nitrate (Fig. 1), a decrease in the content of ammonium in roots (Fig. 2), and no obvious change in the activities of NR and NiR (Figs. 3 and 4). Given the results for the growth of roots (Table 1), we conclude that when soybean seedlings were treated with 1.5 mg/L BPA, the greater uptake of nitrate was not converted to ammonium by the catalysis of NR and NiR. Meanwhile, the decreased ammonium was converted to more proteins, amino acids, or DNA for improving the growth of roots of soybean seedlings. When soybean seedlings were treated with 17.2 mg/L BPA, the nitrate could not be converted to ammonium as effectively (Figs. 1 and 2) because of the decrease in the activities of NR and NiR (Figs. 3 and 4). The content of ammonium originating from the reduction of nitrate was decreased. The increased ammonium (Fig. 2) came mainly from photorespiration, direct absorption, and deamination of N compounds 35. The increased ammonium (Fig. 2) could not be converted well to proteins, amino acids, or DNA. The excess accumulation of ammonium led to the inhibition of the growth of roots of soybean seedlings (Table 1). When the concentration of BPA was increased to 50.0 mg/L, the cellular structure of roots was obviously damaged, leading to a decrease in the ability of roots to absorb nitrate (Fig. 1). The content of ammonium originating from the reduction of nitrate was decreased because of the decrease in the content of nitrate and the activities of NR and NiR (Figs. 3 and 4). The increased ammonium (Fig. 2) came mainly from the deamination of N compounds (including proteins, nucleus acids, or DNA). The excess accumulation of ammonium and decrease in the content of proteins obviously inhibited the growth of roots of soybean seedlings (Table 1). In brief, the change in the activities of NR and NiR and the content of ammonium in roots of soybean seedlings was one reason why the growth of roots was changed and is one of the effect mechanisms of BPA on roots. However, this mechanism was not observed in the recovery period. That is to say, the change in the growth of roots was not correlated with that of the content of ammonium or the activities of NR and NiR. This is a very interesting phenomenon and will be investigated in the future.

The analysis described above indicated that the activities of NR and NiR were important in the change in nitrogen nutrition in plants treated with BPA. The decrease in the activities of NR and NiR might be related to many factors. It has been reported that BPA can induce the accumulation of free radicals in the cell 36. The excess accumulation of free radicals led to the peroxidation of membrane lipid in the cell, the destruction of DNA molecules, and the inhibition in the synthesis of NR and NiR 37, 38. Thus, the activities of NR and NiR were decreased. In addition, the inhibition in the activities of NR has been suggested to be due to a direct interaction between BPA and functional −SH groups of NR 39. The decrease in the activity of NiR might have been associated with the decrease in the nitrite that is a substrate of the reduction reaction catalyzed by NiR. Finally, the decrease in the activities of NR and NiR might also be a combined result of decreased NOmath image uptake or carbon fixation by roots and the low NOmath image translocation in the xylem 40. These hypotheses should be investigated in the future.

CONCLUSIONS

Our results demonstrate that the effects of BPA on the root growth of soybean seedlings depends on the concentration of BPA. Treatment with BPA at a low concentration (1.5 mg/L) caused improvement in the growth of roots, whereas treatment with BPA at higher concentrations (17.2 and 50.0 mg/L) led to inhibition in the growth of roots. In addition, we showed that the effects of BPA on the growth of roots resulted from the change in N nutrition. Further investigations will be required to reveal the details of the effect mechanisms of BPA on plants.

Acknowledgements

The authors are grateful for the financial support of the National Natural Science Foundation of China (31170477), the Natural Science Foundation of Jiangsu Province (BK2011160), and the Independent Research Project of Jiangnan University (JUSRP11110).

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