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Indole-3-acetic acid promotes cadmium (Cd) accumulation in a Cd hyperaccumulator and a non-hyperaccumulator by different physiological responses
Ecotoxicology and Environmental Safety 191 (2020) 110213
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Indole-3-acetic acid promotes cadmium (Cd) accumulation in a Cd hyperaccumulator and a non-hyperaccumulator by different physiological responses
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Jiakang Rana,b, Wen Zhenga, Hongbin Wanga,b,∗, Haijuan Wanga,b, Qinchun Lia,b a b
Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, 650500, China Yunnan Key Lab of Soil Carbon Sequestration and Pollution Control, Kunming, 650500, China
ARTICLE INFO
ABSTRACT
Keywords: Auxin Cadmium Physiological responses Solanum nigrum Solanum melongena
To study the effects of indole-3-acetic acid (IAA) on cadmium (Cd) accumulation and the physiological responses of the Cd hyperaccumulator Solanum nigrum and non-hyperaccumulator Solanum melongena, a pot experiment was conducted in soil containing 2 mg kg−1 Cd in which different concentrations of IAA (0, 10, 20, or 40 mg L−1) were sprayed on plant leaves. The results showed that Cd accumulation in shoots of S. nigrum was significantly increased by 30% after the addition of 10 mg L−1 IAA under 2 mg kg−1 Cd stress compared to that in the control, but shoot Cd accumulation showed no significant change in S. melongena after this IAA treatment. Additionally, the growth and the proline content in the two species were significantly increased by 20 mg L−1 IAA. The activities of peroxidase and catalase in leaves of S. nigrum and the activity of superoxide dismutase (SOD) in S. melongena were significantly increased and their malondialdehyde content was significantly decreased compared to those in the control. The root activity of S. nigrum was significantly improved after 10 and 20 mg L−1 IAA treatments, but no significant difference was observed in S. melongena. The correlation analysis results showed that the Cd concentration in leaves of S. nigrum was significantly and positively correlated with the carotenoid and proline contents, and there was also a significant positive correlation between the Cd concentration and SOD activity in leaves of S. melongena. Therefore, S. nigrum is an ideal plant for the phytoextraction of Cd-contaminated soil assisted by IAA. IAA promotes Cd accumulation in plant shoots by enhancing the accumulation of carotenoids and proline in S. nigrum and maintaining a high leaf SOD activity in S. melongena.
1. Introduction Cadmium (Cd) has become one of the most important metal pollutants in China because of its mobility, long-lasting toxicity, strong chemical activity in the environment and easy accumulation in the food chain (Tang et al., 2016). In China, the area of Cd-contaminated cultivated land exceeds 1.3 × 105 ha, accounting for 1/5 of the total cultivated land; the soil in 25 regions of more than 11 provinces and cities is affected by Cd pollution (Rafiq et al., 2014). Yang et al. (2018) has reported that the average Cd concentration in 1041 agricultural sites in China is 2.9 times higher than the Environmental Quality Standard for Soils in China (Grade II value: 0.3 mg kg−1, which is the
threshold value of Cd in soil to guarantee agricultural production and human health), with a rate exceeding 36.7%, on average. Cadmium pollution in soil causes a decline of the quality of agricultural products, such as rice and vegetables in China, which seriously threatens human health and affects the sustainable development of agriculture (Wang et al., 2015). Therefore, it has become urgent to alleviate the Cd concentration in soil, reduce the accumulation of metals in agricultural products, and ensure the safety and health of ecosystems. Phytoremediation, which utilizes plants to remove or immobilize metals from contaminated soils, has received attention because of its eco-friendly, cost-effective and green advantages compared with those of traditional remediation methods, such as soil replacement, leaching
Abbreviations: ANOVA, analysis of variance; Cd, cadmium; CAT, catalase; EDTA-Na2, disodium edetate; GA3, gibberellic acid 3; HSD, honestly significant difference; IAA, indole-3-acetic acid; MDA, malondialdehyde; NAA, naphthalene acetic acid; PBS, phosphatic buffer solution; PGRs, plant growth regulators; POD, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase ∗ Corresponding author. Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, 650500, China. E-mail addresses: [email protected] (J. Ran), [email protected] (W. Zheng), [email protected] (H. Wang), [email protected] (H. Wang), [email protected] (Q. Li). https://doi.org/10.1016/j.ecoenv.2020.110213 Received 27 July 2019; Received in revised form 20 December 2019; Accepted 12 January 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.
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and the lime improvement method (Sarwar et al., 2016). Some Cd hyperaccumulators have been found around the world, including Noccaea (Thlaspi) caerulescens (Baker and Brooks, 1989), Brassica juncea (Salt et al., 1995) and Viola baoshanensis (Liu et al., 2004), among others. However, these hyperaccumulators tend to have a low biomass, a slow growth rate, and strong regionally limited distributions, which make them difficult to use in practice. Therefore, screening for plants with a strong Cd-accumulating ability and large biomass is the key to remediating Cd-contaminated soil. Solanum nigrum is an ideal plant for the remediation of Cd pollution in soil (Wei et al., 2005). Because of its large biomass, short growth cycle and strong tolerance to Cd stress, many scholars have carried out enhancing phytoextraction studies using S. nigrum, such as colonizing Cd-resistant microorganisms in soil (Gao et al., 2010a; Jiang et al., 2016) and application of citric acid (Gao et al., 2011) and fertilizers (Wei et al., 2010; Yang et al., 2019). Additionally, studies have also addressed the effects of plant growth regulators (PGRs) on the growth and Cd extraction of S. nigrum. For instance, Ji et al. (2015a) sprayed 4 different concentrations (0, 10, 100, 1000 mg L−1) of gibberellic acid 3 (GA3) on the leaf surface of S. nigrum grown in soil containing 1 mg kg−1 Cd and found that the biomass of S. nigrum and the concentration of Cd extracted from a single plant were significantly increased after treatment with 1000 mg L−1 GA3 compared to those of the control, increases of 56% and 72%, respectively. Additionally, Ji et al. (2015b) reported that Cd extraction by S. nigrum was as high as 212 ± 16 μg in a single plant after treatment with 100 mg L−1 indole3-acetic acid (IAA), which represented an increase of 158%, but the related physiological mechanism remains unknown. As the first discovered plant hormone, IAA is widely distributed in plants. Studies have shown that under the lead and zinc stress, 10−10 M IAA can effectively promote the growth of roots and leaves of sunflower, reduce the adverse effects of metals on plants, and increase the extraction of these two metals (Fässler et al., 2010). The antioxidant activities of wheat (Triticum aestivum) under 500 or 1000 μM Cd stress were significantly improved by pretreatment with 500 μM IAA (Agami and Mohamed, 2013). Additionally, the dry weight and fresh weight of Trigonella foenum-graecum growing in soil containing 3 mg kg−1 Cd were also significantly increased by pretreatment with 10 μM IAA (Bashri and Prasad, 2016). Therefore, IAA enhances the ability of plants to resist metal stress and promotes the growth of plants under stress. However, there are few comparative reports on the accumulation of Cd and the physiological and biochemical changes in Cd hyperaccumulators and non-hyperaccumulators after exogenous application of IAA. Therefore, we hypothesized that exogenous IAA could result in different physiological and biochemical mechanisms in plants with different Cd-accumulating abilities during the process of Cd accumulation. To test this hypothesis, a pot experiment was conducted to determine the biomass, Cd accumulation and physiological responses of the Cd hyperaccumulator S. nigrum and the non-hyperaccumulator Solanum melongena after they were exposed to 2 mg kg−1 Cd in soil and had different concentrations of IAA (0–40 mg L−1) sprayed on the surface of their leaves. Our results will provide a scientific basis for a phytohormone-based technology for the phytoremediation of metalcontaminated soil.
non-hyperaccumulator S. melongena were purchased at the Longjie Farmers’ Market of Chenggong County, Kunming City and were cultivated in clean soil in a greenhouse. After 4–5 leaves (8–10 cm height) of S. nigrum developed, seedlings of S. melongena with good growth and uniform size (with 3–4 leaves, 15–17 cm height) were selected for the pot experiment. The tested soil was collected from the pear garden of KUST and was air-dried and sieved with a 5 mm sieve. The tested soil was fully mixed according to the ratio of soil: river sand: humus = 2:1:1 and was treated with N, P, and K nutrients as the base fertilizer (N: P2O5: K2O = 0.15: 0.10: 0.15 g kg−1 soil, dry weight). The soil pH was 7.09 ± 0.02, and the contents of organic matter, total nitrogen, total phosphorus, and available potassium in the soil were 69.2 ± 1.4, 2.64 ± 0.01, 0.895 ± 0.007 and 0.22 ± 0.01 g kg−1, respectively. The concentrations of Pb, Zn and Cu in the soil were 44.39 ± 0.88, 77.35 ± 4.52 and 155.60 ± 4.29 mg kg−1, respectively, but Cd and As were below the detection limit (Cd: 0.006 mg kg−1; As: 0.011 mg kg−1). Based on the “Soil Environmental Quality: Risk Control Standard for Soil Contamination of Agricultural land” in China, the concentrations of Pb, Zn and Cu in the tested soil were all lower than the risk screening values for soil contamination of agriculture land, which are 120, 250 and 200 mg kg−1, respectively, when the soil pH ranges from 6.5 to 7.5. 2.2. Experimental design According to the results of a pre-experiment, the concentration of Cd was designed to be 2 mg kg−1, which was added in the form of CdCl2·2.5H2O. After 4 weeks of stabilization, each pot was filled with 1 kg soil. The 24 pots were randomly arranged in a randomized block design (four blocks, each block containing 3 pots for one plant) in a greenhouse. The IAA concentrations were designed to be 0, 10, 20 and 40 mg L−1, and each treatment was replicated three times. The two plant species were planted separately, and ten individuals of S. nigrum were planted in each pot. Due to the large biomass of S. melongena, four individuals were planted in each pot to ensure that the growth and biomass of the plants in each pot were similar. The plants were cultured in a greenhouse under natural light with the temperature varying from 15 to 25 °C (night/day). After 30 days, the leaf surfaces were sprayed with IAA. To guarantee the consistency of each treatment, the volume of IAA sprayed on the plant leaves in each pot was 25 mL, and three pots (replicates) were included in each IAA treatment. The control group replaced IAA with 25 mL deionized water. Due to the adaptation of the plants to IAA, they were sprayed every three days for the first two weeks and then sprayed once every seven days for the next two weeks. Spraying occurred seven times. After being sprayed with IAA the first time, the two plant species were grown for 30 days and harvested. The plant surfaces were cleaned with tap water, and the plants were divided into two parts. One part of the fresh samples was rinsed twice with deionized water, immediately frozen with liquid nitrogen, and then placed in self-sealing bags and stored at −18 °C in a refrigerator to prepare for the determination of physiological changes. The other part of the fresh samples was divided into roots, stems and leaves, and the roots of the plants were soaked in 20 mM disodium edetate (EDTA-Na2) for 15 min to remove residual Cd from the root surface. Then, the roots, stems and leaves were washed twice with deionized water. The cleaned plant materials were dried in an oven at 105 °C for 30 min and then dried at 75 °C to a constant weight for the determination of the Cd concentration.
2. Materials and methods 2.1. Plant materials and soil
2.3. Analysis methods
Seeds of the Cd hyperaccumulator S. nigrum were collected from the campus of Kunming University of Science and Technology (KUST) in July 2016. After being soaked for 10–12 h, they were disinfected with 0.2% NaClO for 10 min and were then washed with running water and deionized water several times. Then, the seeds were dried with filter paper and were spread in clean soil for germination. Seedlings of the
The soil was digested with aqua regia-HClO4, and plant samples were digested with HNO3–H2O2 for the determination of the Cd concentration. A soil sample (0.2 g) that had been passed through a 100 mesh sieve was added to 5 mL aqua regia and soaked for 12 h and was 2
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Fig. 1. Effects of IAA on biomass and height of two plants treated by 2 mg kg−1 Cd. Data are expressed as the mean value ± standard error of three replicates in each treatment (n = 3). The different letters indicate that the fresh weight and plant height have a significant difference among different concentrations of IAA treatments (P < 0.05).
heated and digested at 80 °C for 30 min, 120 °C for 60 min and 140 °C for 60 min in sequence. After grinding, the dried plant materials (0.2 g) were added to 5 mL HNO3 and heated as described above. After cooling, 1 mL of HClO4 and 1 mL 30% H2O2 were added to the soil and plant samples, which were then sequentially heated at 140 °C for 60 min. The digested samples were cooled to 25 mL, filtered, bottled and examined using an atomic absorption spectrometer (Varian AA240FS, USA). The standard materials (GBW-10048) and standard solution of Cd (GSB 041721-2004) were purchased from the National Research Centre for Certified Reference Materials. The working curve for Cd determination was y = 0.2569x+0.0021, R2 = 0.9997, where y is the absorbance value and x is the Cd concentrations (mg·L−1). The recovery of Cd was 95–98%, which met the quality control requirements for Cd determination. The root morphologies of S. nigrum and S. melongena were scanned using a scanner (HP LaserJet M 1005 MEP), and the relevant indicators, including the root length, root tip number and root surface area, were analysed using the Winrhizo root system analysis software (He et al., 2009). The root activity, which mainly reflects the absorption, synthesis, oxidation and reduction abilities of roots, was determined by the triphenyltetrazolium chloride (TTC) method (Zhang and Qu, 2003), and the ATPase activity of the plasma membrane in root cells was determined as described by Forbush (1983). Plant leaves (2.0 g) were collected and randomly mixed from each individual plant in each pot. The leaves were added to a phosphate buffer solution (PBS, pH = 7.8), homogenized in an ice bath and centrifuged at 15,000×g, 20 °C for 20 min. The supernatant was stored at 4 °C for the determination of the enzyme activities and MDA contents. The activity of superoxide dismutase (SOD) was analysed by the nitrogen blue tetrazolium method (Beyer and Fridovich, 1987), peroxidase (POD) activity was determined by the guaiacol method and catalase (CAT) activity was determined using potassium permanganate titration (Aebi, 1984; Beffa et al., 1990). The content of malondialdehyde (MDA) was determined by the thiobarbituric acid method (Heath and Packer, 1968). Additionally, the chlorophyll contents were determined by the 95% ethanol extraction method (Sartory and Grobbelaar, 1984), and the proline assay was followed by the acid ninhydrin method (Lee and Takahashi, 1966).
2.4. Statistical analysis Data were analysed by one-way and two-way analysis of variance (ANOVA) by using the statistical analysis system software (SAS 9.2), and multiple comparisons were performed using Tukey's HSD (honestly significant difference) method. The purpose of one-way ANOVA was to determine whether a significant difference existed for a given parameter among the 4 IAA treatments. There were three degree of freedom (DF) in one-way ANOVA: The DF among groups was 3 (ν1), the DF within groups was 8 (ν2) and the total DF was 11 (ν3); when ν1 = 3 and ν2 = 8, F0.05 = 4.07 and F0.01 = 7.59. Because two factors, IAA and plant species, were included in the present study, we also used two-way ANOVA to determine whether a significant difference existed for a given parameter between the plant species (Factor A), among the IAA concentrations (factor B) and for the interaction between plant species and IAA (factor A × B). There were five degree of freedom (DF) in two-way ANOVA: The DF of plants was 1 (ν1), the DF of IAA was 3 (ν2), the DF of plants × IAA was 3 (ν3), the DF of error was 16 (ν4) and the total DF was 23 (ν5). When ν1 = 1 and ν4 = 16, F0.05 = 4.49 and F0.01 = 8.53; when ν2 = ν3 = 3 and ν4 = 16, F0.05 = 3.24 and F0.01 = 5.29 (Supplementary Table 1). To compare the Cd accumulation and translocation ability between the two plants, the bioconcentration factor (BCF) and translocation factor (TF) were calculated. The BCF is defined as the ratio of the Cd concentration in shoots to that in soil, and the TF is defined as the ratio of the Cd concentration in shoots to that in roots. The level of significant difference was set at α = 0.05, and the level of extremely significant difference was set at α = 0.01. Stepwise regression analysis was performed by the statistical product and service solutions (SPSS 20.0) software, and the figures were drawn using Origin 9.0 software. 3. Results 3.1. Effect of IAA on plant growth The fresh weights of both plants were significantly higher than that of the control at the two lowest IAA treatments (10 and 20 mg L−1, P < 0.05, F = 9.575, 14.756), with maximums of 15.60 and 41.93 g 3
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Fig. 2. Effects of IAA on Cd accumulation by two plants treated by 2 mg kg−1 Cd.
plant−1, respectively, when treated with 10 mg L−1 IAA (Fig. 1a). No significant difference in the fresh weight of the two plants was observed between the 0 and 40 mg L−1 IAA treatments. The heights of both plants were significantly increased after the 20 mg L−1 IAA treatment compared to that of the control (P < 0.05, F = 10.806, 22.877, Fig. 1b), but a low concentration of IAA (10 mg L−1 IAA) significantly increased the height of S. nigrum (P < 0.05, F = 10.806).
than those in the control. However, the Cd concentrations in the shoots and roots of S. melongena were significantly increased only after the 20 mg L−1 IAA treatment compared to those in the control, and a significant decrease was observed after the highest IAA treatment (40 mg L−1, P < 0.05, F = 17.979, 12.859, Fig. 2a). In the case of BCFs, a significant increase in S. melongena was only observed after the 20 mg L−1 IAA treatment (P < 0.05, F = 17.919). For TFs, no significant changes were observed in the two plants with the addition of IAA (P > 0.05, F = 3.836, 1.173, Fig. 2c).
3.2. Effect of IAA on Cd accumulation in plants The addition of different concentrations of IAA promoted the accumulation of Cd by S. nigrum, especially after the 10 mg L−1 IAA treatment, up to 49.52 mg kg−1 in its shoots with an increasing rate of 30%. With the increase in the IAA concentration, the Cd concentrations in the roots, shoots (P < 0.05, F = 8.251, 56.994, Fig. 2a) and BCFs (P < 0.01, F = 50.254, Fig. 2b) of S. nigrum were significantly higher
3.3. Effects of IAA on the physiological and biochemical responses of plants 3.3.1. Root morphology, root activity and ATPase activity of plasma membranes IAA significantly increased the total root length, root surface area 4
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Fig. 3. Effect of IAA on root morphology of two plants treated by 2 mg kg−1 Cd.
and root tip number of S. nigrum (P < 0.05, F = 7.023–15.842, Fig. 3) at 20 mg L−1 IAA, but no significant differences were observed after the other treatments compared to those of the control. In addition, the total root length, root surface area and root tip number of S. melongena were increased significantly at the highest IAA treatment compared to those of the control (P > 0.05, F = 7.588–7.239), but no significant differences were observed between the 10 and 20 mg L−1 IAA treatments (Fig. 3). Compared to that of the control, the root activity of S. nigrum was significantly increased with the addition of IAA (P < 0.05, F = 29.394), but the root activity of S. melongena was significantly increased only after treatment with a high concentration of IAA (P < 0.05, F = 7.774). No significant difference in root activity was observed in S. melongena after the other IAA treatments (Fig. 4a). The ATPase activity of the plasma membrane from root cells of S. nigrum and S. melongena were significantly increased when treated with low and high concretions of IAA (P < 0.05, F = 22.537, 8.932, Fig. 4b), but no significant difference was observed for the other IAA treatments compared to the control.
3.3.2. Effect of IAA on the activities of antioxidant enzymes and the proline and MDA contents of plants The activities of antioxidant enzymes in the leaves of plants varied with the addition of different concentrations of IAA. The SOD activity in the leaves of S. nigrum was significantly increased compared to that in the control when treated with 10 mg L−1 IAA (P < 0.05, F = 134.023, Fig. 5a), but the SOD and CAT activities in this plant were significantly decreased when treated with a high concentration of IAA (P < 0.05, F = 134.023, 51.474, Fig. 5a, c). In the case of S. melongena, the POD and CAT activities were also significantly increased after treatment with a high concentration of IAA (P < 0.05, F = 15.31, 42.551), but a significant increase in SOD activity was only observed after the 20 mg L−1 IAA treatment (P < 0.05, F = 40.342, Fig. 5). With the increasing concentrations of IAA, the content of malondialdehyde (MDA) in leaves of the plants was significantly decreased (P < 0.05, F = 9.583, 20.626, Fig. 5d). When leaves were sprayed with 20 mg L−1 IAA, the MDA content in the leaves of S. nigrum reached the minimum value (0.015 μmol g −1). For S. melongena, the MDA content in the leaves of this plant was significantly decreased compared to that in the control, except after the 10 mg L−1 IAA treatment 5
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Fig. 4. Effect of IAA on root activity and ATPase activity of plasma membrane in root cells treated by 2 mg kg−1 Cd.
(Fig. 5d). The proline contents of the plants were significantly increased after the two lowest IAA treatments compared to that in the control (P < 0.05, F = 24.778, 17.7, Fig. 5e). However, the proline contents of the two plants were not significantly different from that of the control when sprayed with a high concentration of IAA (P > 0.05, Fig. 5e).
significant negative correlation with CAT (X9) and chlorophyll a (X11). The regression equation for S. melongena was Y = 6.391 + 1.75 E005X7-0.349X9-1.959X11 (P < 0.01, r = 0.964, F = 35.188).
3.3.3. Effect of IAA on the contents of photosynthetic pigments As shown in Table 1, the contents of photosynthetic pigments (chlorophyll a, b and carotenoids) in the two plants were significantly higher than those of the control (P < 0.05, F = 4.799, 14.778) when treated with 10 mg L−1 IAA. There was no significant difference among the other IAA treatments compared to the control. The results of twoway analysis of variance (ANOVA) showed that the contents of photosynthetic pigments in plants were significantly affected by the concentration of IAA (P < 0.01, Table 2). The results of two-way ANOVA showed that plant height, Cd accumulation, root surface area, root tip number, root activity, ATPase activity of plasma membrane in root cells, SOD and POD activities, and MDA content were significantly affected by the plant species, IAA concentration and their interactions (P < 0.01, Figs. 1–5).
Cadmium is toxic to plant growth, development and reproduction. Plant growth is slower when the Cd concentration in the growth medium increases. When present in soil at a high concentration, Cd destroys the chloroplast structure of plants, leading to yellowing or necrosis (Shi et al., 2010). The Cd concentration used in this study was 2 mg kg−1 according to our previous experiment because no visible toxicity symptoms were observed in two tested plants at this Cd concentration. It was also reported that after treatment with 2 mg kg−1 Cd, the biomasses of the aboveground and underground parts of S. nigrum were not significantly different from those of the control (Gao et al., 2010; Jiao et al., 2013). Moreover, it was found that when the Cd concentration of soil was 2 mg kg−1, the 13 varieties of S. melongena studied essentially grew normally and showed no symptoms, such as chlorosis or green deficiency, indicating that the two plants could grew well and had high tolerance to Cd at this Cd concentration. However, the Cd tolerance of S. nigrum was stronger than that of S. melongena. Wei et al. (2005) planted S. nigrum in soil containing 0, 10, 25, 50, 100 and 200 mg kg−1 Cd and found that neither the average height nor the shoot dry mass of S. nigrum was reduced significantly after the 10 and 25 mg kg−1 Cd treatments, respectively. Nevertheless, the average height of S. nigrum was significantly reduced (P < 0.05) after the 50, 100 and 200 mg kg−1 Cd treatments. Comparatively, the tolerance of S. melongena for Cd was weak and its growth was significantly reduced by 3 mg kg−1 Cd (Singh and Prasad, 2014). As a plant growth regulator, IAA is widely used to increase the growth of plants (Zhu et al., 2013). Khan et al. (2019) reported that compared to Cd treatment alone, the fresh weights of root and shoots in tomato seedlings were significantly increased by 16.3% and 11.17% after adding 5 μM IAA under 100 μM Cd stress, respectively. In the present study, application of the two lowest IAA concentrations was associated with a significant increase in fresh weight of both plants, and the shoot Cd concentration in S. nigrum increased (Fig. 1a). This result indicated that IAA could promote the growth of the hyperaccumulator
4. Discussion 4.1. Plant growth and tolerance to Cd are improved by IAA
3.4. Stepwise regression analysis A multiple stepwise regression analysis was performed to test the correlation among the different indexes. Here, Y is the Cd concentration in the leaves of plant, X1 is the IAA treatment concentration, X2 is the total root length, X3 is the root surface area, X4 is the root tip number, X5 is the root activity, X6 is the ATPase activity of the plasma membrane in root cells, X7 is the SOD activity, X8 is the POD activity, X9 is the CAT activity, X10 is the MDA content, X11 is chlorophyll a, X12 is chlorophyll b, X13 is carotenoid and X14 is the proline content. The regression equation for S. nigrum was Y = 11.705–12.369X11+86.191X13+1.059X14 (P < 0.01, r = 0.947, F = 23.008). Therefore, the Cd concentration in leaves of S. nigrum was significantly and positively correlated with carotenoids (X13) and the proline content (X14) and negatively correlated with chlorophyll a (X11). There was a significant positive correlation between the Cd concentration and SOD activity (X7) in leaves of S. melongena and a 6
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Fig. 5. Effect of IAA on activities of antioxidative enzymes, malondialdehyde (MDA) and proline contents in plants treated by 2 mg kg−1 Cd.
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Table 1 Effect of IAA on the photosynthetic pigment contents of plants at 2 mg kg−1 Cd treatment. Plant species
IAA concentration (mg L−1)
S. nigrum
0 10 20 40 0 10 20 40
S.melongena
Photosynthetic pigment contents (mg·g−1) Chlorophyll a 1.03 ± 0.13 b 1.43 ± 0.05a 1.11 ± 0.14 ab 1.15 ± 0.19 ab 1.11 ± 0.02 b 1.22 ± 0.03a 1.14 ± 0.02 b 1.21 ± 0.02a
Chlorophyll b 0.46 ± 0.05 b 0.71 ± 0.08a 0.55 ± 0.08 ab 0.51 ± 0.09 ab 0.45 ± 0.02 b 0.56 ± 0.04a 0.51 ± 0.02 ab 0.52 ± 0.02 ab
Carotenoid 0.22 ± 0.03 b 0.30 ± 0.02a 0.23 ± 0.03 b 0.26 ± 0.01 ab 0.24 ± 0.01 b 0.29 ± 0.03a 0.25 ± 0.01 ab 0.28 ± 0.02a
Total photosynthetic pigments 1.49 ± 0.18 b 2.15 ± 0.22a 1.66 ± 0.22 ab 1.67 ± 0.28 ab 1.56 ± 0.08 b 1.78 ± 0.07a 1.66 ± 0.08 ab 1.72 ± 0.06 ab
Data are expressed as the mean value ± standard error of three replicates in each treatment (n = 3). The different letters in the column of each index indicate that there is a significant difference among different concentrations of IAA treatments in a plant species (P < 0.05).
S. nigrum and enhance its tolerance to Cd. On the other hand, the heights of the two plants varied after administration of IAA (Fig. 1b), possibly due to the difference in plant species, which makes plant tissue sensitive to IAA. Ji et al. (2015) found that IAA can effectively increase the biomass and Cd concentration of shoots in S. nigrum. In our study, the shoot Cd concentration in S. nigrum was significantly improved after IAA application. However, this obvious shoot Cd enhancement was only observed after the 20 mg L−1 IAA treatment in S. melongena (Fig. 2a). This result indicates that the Cd accumulation in shoots of S. nigrum is enhanced by a low concentration of IAA, although its TF values showed no significant change (Fig. 2c). Compared to S. nigrum, the accumulation of Cd in S. melongena was enhanced by IAA at a higher level.
was also markedly increased (Fig. 3), indicating that IAA could improve the root morphology of the Cd hyperaccumulator by promoting the division and differentiation of plant cells, which may be beneficial to Cd accumulation. Additionally, the root morphology of S. melongena was also significantly improved after the highest IAA treatment (Fig. 3). Root activity is a physiological indicator that can be used to characterize plant root growth and stress resistance. In this study, the root development of S. nigrum was notably promoted by application of IAA. Accordingly, the root activity of S. nigrum was improved by application of IAA, but an obvious increase in root activity in S. melongena was only shown after the highest IAA treatment (Fig. 4a). This result indicated that the root activity of S. nigrum was facilitated by low concentrations of IAA, which in turn, contributed to plant growth. Additionally, the ATPase activity of the plasma membrane in root cells in S. nigrum and S. melongena was obviously increased when treated with 10 and 40 mg L−1 IAA, respectively (Fig. 4b). It has been documented that exogenous application of IAA significantly increases H+-ATPase activity in the plasma membrane in Al-stressed alfalfa roots and promotes H+ secretion from root tips (Wang et al., 2017). The decreasing pH value in the rhizospheric environment changes the bioavailability and mobilization of Cd and affects Cd uptake by plants.
4.2. IAA improves the root morphology of plants Generally, the root system is the first organ to respond to stress, and plants adapt to environmental stress by changing their root morphology and distribution (Takahashi and Asada, 1983). Qin et al. (2018) reported that the root length, root volume, root dry matter weight, root effective surface area and total root area of winter wheat decreased with an increasing Cd concentration. However, IAA treatment promotes plant cell division, root growth and root surface areas. The root growth of sunflowers was effectively promoted by 3 or 6 mg L−1 IAA (Liphadzi et al., 2006). Our results showed that the total root length, root surface areas and root tip numbers of S. nigrum were obviously increased with the addition of 20 mg L−1 IAA, and the Cd concentration in S. nigrum
4.3. IAA promotes Cd accumulation in the two plants by different physiological mechanisms Superoxide dismutase (SOD) is an important protective enzyme in plants. Under normal conditions, the activity of SOD in plants is not high,
Table 2 Two-way ANOVA for photosynthetic pigment contents. Index
Source
Degree of freedom
Sum of squares
Mean square
F ratio
F0.05
F0.01
P value
Chlorophyll a
Plant IAA Plant Error Total Plant IAA Plant Error Total Plant IAA Plant Error Total Plant IAA Plant Error Total
1 3 3 16 23 1 3 3 16 23 1 3 3 16 23 1 3 3 16 23
0.001 0.225 0.081 0.16
0.001 0.075 0.027 0.01
0.093 7.439** 2.729
4.49 3.24 3.24
8.53 5.29 5.29
0.764 0.002** 0.078
0.013 0.099 0.021 0.048
0.013 0.033 0.007 0.003
3.753 9.947** 2.165
4.49 3.24 3.24
8.53 5.29 5.29
0.071 0.001** 0.132
0.001 0.018 0.003 0.0096
0.001 0.006 0.001 0.0005
1.657 13.212** 1.212
4.49 3.24 3.24
8.53 5.29 5.29
0.216 0.000** 0.337
0.023 0.618 0.183 0.448
0.023 0.206 0.061 0.028
0.807 7.288** 2.148
4.49 3.24 3.24
8.53 5.29 5.29
0.382 0.003** 0.134
Chlorophyll b
Carotenoid
Total photosynthetic pigments
× IAA
× IAA
× IAA
× IAA
The double asterisk (**) shows the difference is very significant (P < 0.01). 8
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but it is increased to eliminate peroxides in plants under adverse conditions, thus maintaining the normal metabolism of plants (Tian et al., 2012). Peroxidase protects cells from damage by removing excess lipid peroxidation products from the body (Doğanlar, 2013). Catalase is a catalyst that hydrolyses H2O2 into H2O and O2, thereby avoiding the risk of peroxidation in membrane lipids. Exogenous IAA can resist metal stress by regulating the activities of antioxidant enzymes (SOD, POD and CAT etc.) in plants (Bashri and Prasad, 2016). In this study, the activities of POD and CAT in S. nigrum and the activity of SOD in S. melongena were notably increased by 20 mg L−1 IAA (Fig. 5), indicating that the ability of S. nigrum to remove reactive oxygen species (ROS) was stronger than that of S. melongena after the addition of IAA. The stepwise regression analysis results showed that the Cd concentration in leaves of S. melongena was significantly and positively correlated with SOD activity and negatively correlated with CAT activity. Malondialdehyde is a peroxidative product of membrane lipids in plant cells, and its content reflects the level of membranous peroxidation in plants to some extent. The level of MDA in leaves of T. foenum-graecum was significantly reduced after a 10 μM IAA treatment under different concentrations of Cd stress (Bashri and Prasad, 2016). Our study found that the MDA content in both plants obviously decreased after IAA application, but after the treatment with 20 mg L−1 IAA, the MDA content in leaves of S. nigrum was much lower than that in S. melongena (Fig. 5d). This result indicates that the addition of IAA reduced the peroxidation degree of membrane lipids in the two plants and increased their tolerance to Cd. However, compared to S. melongena, S. nigrum had a more effective defence system to decrease the peroxidation of membrane lipids. The contents of chlorophyll in plants can be used as an indicator to measure the photosynthetic capacity and the damage degree of plants in response to stress. It has been reported that the application of IAA to Chlorella vulgaris has a beneficial effect on the photosynthetic apparatus under metal stress (Piotrowska-Niczyporuk et al., 2012). In the present study, the contents of photosynthetic pigments in the leaves of the two plants were markedly increased when treated with 10 mg L−1 IAA (Table 1). This result indicated that treatment with a suitable concentration of IAA could promote photosynthesis in S. nigrum and S. melongena, which is beneficial to the accumulation of photosynthetic pigments in the leaves of plants, but this promotion effect on S. nigrum was more obvious (Table 1). Stepwise regression analysis showed that the Cd concentration in the leaves of the plants was significantly and negatively correlated with the chlorophyll a content, probably due to the inhibition of chlorophyll biosynthesis by Cd (Singh and Prasad, 2015) and because chlorophyll a is more sensitive than other photosynthetic pigments. Additionally, the Cd concentration in the leaves of S. nigrum was significantly and positively correlated with carotenoids, probably because carotenoids can protect photosynthesis under stress by removing singlet oxygen (Singh and Prasad, 2015), thereby increasing plant biomass and promoting Cd accumulation by S. nigrum. Additionally, free proline is an osmotic adjustment substance in plants, and its content can reflect the stress resistance of plants to some extent. Proline can form a nontoxic proline-metal complex by chelation, thereby enhancing plant tolerance to stress (Sun et al., 2007). In the present study, the proline content of the plants was obviously increased after the lowest two IAA treatments, but the increase in S. nigrum was higher than that in S. melongena (Fig. 5e). The stepwise regression analysis results also showed that the Cd concentration in leaves of S. nigrum was significantly and positively correlated with the proline content. Therefore, IAA can enhance the resistance of S. nigrum by adjusting its proline level. On the whole, exogenous addition of IAA could promote the accumulation of Cd in leaves by promoting physiological indicators, which were different in the Cd hyperaccumulator and non-hyperaccumulator.
synthetic PGRs are stable and cheap. Therefore, PGRs are widely applied in agricultural production. During our experimental design, we did not realize the difference between native IAA and synthetic Dicamba or NAA (naphthalene acetic acid), and native IAA is popularly used in some references (Fässler et al., 2010; Agami and Mohamed, 2013; Singh and Prasad, 2015). We will consider the difference of these two phytohormones from different sources in the future, and a comparative experiment is needed to determine the effects of native IAA and synthetic Dicamba/NAA at the same concentration on plant Cd uptake and physiological responses. Regarding the efficiency of IAA application under field conditions, little information is available on the phytoremediation efficiency of metals in soil because many studies have focused on pot experiments, such as those of Liphadzi et al. (2006), Ji et al. (2015b) and Bashri and Prasad (2016). However, IAA is successfully used in the field to alleviate salinity stress in maize (Kaya et al., 2013) and improve the net photosynthetic rate of Zizania latifolia (Li et al., 2019). At present, we are conducting a field experiment in Cd–Pb–As combined polluted soil in the suburb of Gejiu City, Yunnan Province to examine the Cd phytoextraction efficiency of Solanum nigrum by IAA or/and kinetin application. 5. Conclusions Our work indicated that under the stress of 2 mg kg−1 Cd, the growth of S. nigrum and S. melongena was significantly increased by the addition of 20 mg L−1 IAA, which also improved the osmotic adjustment and activities of their antioxidant enzymes and alleviated the lipid peroxidation of cell membranes. The shoot Cd concentration in S. nigrum was significantly increased with the application of 10–40 mg L−1 IAA, but this significant increase of shoot Cd was only observed at 20 mg L−1 IAA in S. melongena. Furthermore, the Cd concentration in shoots of S. nigrum was significantly and positively correlated with the contents of carotenoid and proline, and there was a significant positive correlation between the Cd concentration and SOD activity in leaves of S. melongena. Therefore, exogenous addition of IAA could enhance the accumulation of Cd in shoots by promoting physiological indicators, which were different in the Cd-hyperaccumulator and non-hyperaccumulator. It should also be noted that with the addition of IAA, S. nigrum showed a stronger ability to accumulate Cd in shoots compared to that in S. melongena. Compared to chelating agent-induced phytoextraction technology, native phytohormones or other PGRs are widely used in agricultural production. IAA is easily absorbed by plants and is degraded in plants by enzymatic reactions. Therefore, a significant negative effect on soil quality may be avoided if a suitable concentration of IAA is added. However, our present work was conducted in a greenhouse with a pot experiment, which cannot fully reflect the actual situation in the field. Therefore, this IAA-assisted Cd phytoextraction should be further tested in the field during the phytoremediation of Cdcontaminated soil. CRediT authorship contribution statement Jiakang Ran: Investigation, Writing - original draft, Writing - review & editing. Wen Zheng: Investigation, Data curation, Formal analysis. Hongbin Wang: Conceptualization, Methodology, Writing original draft, Writing - review & editing. Haijuan Wang: Conceptualization, Methodology, Writing - review & editing. Qinchun Li: Formal analysis, Writing - review & editing.
4.4. Potential of IAA-induced phytoextraction technology in practice
Acknowledgments
It should be noted that we used native auxin in our experiment, and it is easily degraded in the natural environment. Comparatively,
This work was supported by the Key Technologies Research and Demonstration Project for Yunnan's Soil Remediation (2018BC004–2). 9
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Appendix A. Supplementary data
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Indole-3-acetic acid promotes cadmium (Cd) accumulation in a Cd hyperaccumulator and a non-hyperaccumulator by different physiological responses
T
Jiakang Rana,b, Wen Zhenga, Hongbin Wanga,b,∗, Haijuan Wanga,b, Qinchun Lia,b a b
Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, 650500, China Yunnan Key Lab of Soil Carbon Sequestration and Pollution Control, Kunming, 650500, China
ARTICLE INFO
ABSTRACT
Keywords: Auxin Cadmium Physiological responses Solanum nigrum Solanum melongena
To study the effects of indole-3-acetic acid (IAA) on cadmium (Cd) accumulation and the physiological responses of the Cd hyperaccumulator Solanum nigrum and non-hyperaccumulator Solanum melongena, a pot experiment was conducted in soil containing 2 mg kg−1 Cd in which different concentrations of IAA (0, 10, 20, or 40 mg L−1) were sprayed on plant leaves. The results showed that Cd accumulation in shoots of S. nigrum was significantly increased by 30% after the addition of 10 mg L−1 IAA under 2 mg kg−1 Cd stress compared to that in the control, but shoot Cd accumulation showed no significant change in S. melongena after this IAA treatment. Additionally, the growth and the proline content in the two species were significantly increased by 20 mg L−1 IAA. The activities of peroxidase and catalase in leaves of S. nigrum and the activity of superoxide dismutase (SOD) in S. melongena were significantly increased and their malondialdehyde content was significantly decreased compared to those in the control. The root activity of S. nigrum was significantly improved after 10 and 20 mg L−1 IAA treatments, but no significant difference was observed in S. melongena. The correlation analysis results showed that the Cd concentration in leaves of S. nigrum was significantly and positively correlated with the carotenoid and proline contents, and there was also a significant positive correlation between the Cd concentration and SOD activity in leaves of S. melongena. Therefore, S. nigrum is an ideal plant for the phytoextraction of Cd-contaminated soil assisted by IAA. IAA promotes Cd accumulation in plant shoots by enhancing the accumulation of carotenoids and proline in S. nigrum and maintaining a high leaf SOD activity in S. melongena.
1. Introduction Cadmium (Cd) has become one of the most important metal pollutants in China because of its mobility, long-lasting toxicity, strong chemical activity in the environment and easy accumulation in the food chain (Tang et al., 2016). In China, the area of Cd-contaminated cultivated land exceeds 1.3 × 105 ha, accounting for 1/5 of the total cultivated land; the soil in 25 regions of more than 11 provinces and cities is affected by Cd pollution (Rafiq et al., 2014). Yang et al. (2018) has reported that the average Cd concentration in 1041 agricultural sites in China is 2.9 times higher than the Environmental Quality Standard for Soils in China (Grade II value: 0.3 mg kg−1, which is the
threshold value of Cd in soil to guarantee agricultural production and human health), with a rate exceeding 36.7%, on average. Cadmium pollution in soil causes a decline of the quality of agricultural products, such as rice and vegetables in China, which seriously threatens human health and affects the sustainable development of agriculture (Wang et al., 2015). Therefore, it has become urgent to alleviate the Cd concentration in soil, reduce the accumulation of metals in agricultural products, and ensure the safety and health of ecosystems. Phytoremediation, which utilizes plants to remove or immobilize metals from contaminated soils, has received attention because of its eco-friendly, cost-effective and green advantages compared with those of traditional remediation methods, such as soil replacement, leaching
Abbreviations: ANOVA, analysis of variance; Cd, cadmium; CAT, catalase; EDTA-Na2, disodium edetate; GA3, gibberellic acid 3; HSD, honestly significant difference; IAA, indole-3-acetic acid; MDA, malondialdehyde; NAA, naphthalene acetic acid; PBS, phosphatic buffer solution; PGRs, plant growth regulators; POD, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase ∗ Corresponding author. Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, 650500, China. E-mail addresses: [email protected] (J. Ran), [email protected] (W. Zheng), [email protected] (H. Wang), [email protected] (H. Wang), [email protected] (Q. Li). https://doi.org/10.1016/j.ecoenv.2020.110213 Received 27 July 2019; Received in revised form 20 December 2019; Accepted 12 January 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.
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and the lime improvement method (Sarwar et al., 2016). Some Cd hyperaccumulators have been found around the world, including Noccaea (Thlaspi) caerulescens (Baker and Brooks, 1989), Brassica juncea (Salt et al., 1995) and Viola baoshanensis (Liu et al., 2004), among others. However, these hyperaccumulators tend to have a low biomass, a slow growth rate, and strong regionally limited distributions, which make them difficult to use in practice. Therefore, screening for plants with a strong Cd-accumulating ability and large biomass is the key to remediating Cd-contaminated soil. Solanum nigrum is an ideal plant for the remediation of Cd pollution in soil (Wei et al., 2005). Because of its large biomass, short growth cycle and strong tolerance to Cd stress, many scholars have carried out enhancing phytoextraction studies using S. nigrum, such as colonizing Cd-resistant microorganisms in soil (Gao et al., 2010a; Jiang et al., 2016) and application of citric acid (Gao et al., 2011) and fertilizers (Wei et al., 2010; Yang et al., 2019). Additionally, studies have also addressed the effects of plant growth regulators (PGRs) on the growth and Cd extraction of S. nigrum. For instance, Ji et al. (2015a) sprayed 4 different concentrations (0, 10, 100, 1000 mg L−1) of gibberellic acid 3 (GA3) on the leaf surface of S. nigrum grown in soil containing 1 mg kg−1 Cd and found that the biomass of S. nigrum and the concentration of Cd extracted from a single plant were significantly increased after treatment with 1000 mg L−1 GA3 compared to those of the control, increases of 56% and 72%, respectively. Additionally, Ji et al. (2015b) reported that Cd extraction by S. nigrum was as high as 212 ± 16 μg in a single plant after treatment with 100 mg L−1 indole3-acetic acid (IAA), which represented an increase of 158%, but the related physiological mechanism remains unknown. As the first discovered plant hormone, IAA is widely distributed in plants. Studies have shown that under the lead and zinc stress, 10−10 M IAA can effectively promote the growth of roots and leaves of sunflower, reduce the adverse effects of metals on plants, and increase the extraction of these two metals (Fässler et al., 2010). The antioxidant activities of wheat (Triticum aestivum) under 500 or 1000 μM Cd stress were significantly improved by pretreatment with 500 μM IAA (Agami and Mohamed, 2013). Additionally, the dry weight and fresh weight of Trigonella foenum-graecum growing in soil containing 3 mg kg−1 Cd were also significantly increased by pretreatment with 10 μM IAA (Bashri and Prasad, 2016). Therefore, IAA enhances the ability of plants to resist metal stress and promotes the growth of plants under stress. However, there are few comparative reports on the accumulation of Cd and the physiological and biochemical changes in Cd hyperaccumulators and non-hyperaccumulators after exogenous application of IAA. Therefore, we hypothesized that exogenous IAA could result in different physiological and biochemical mechanisms in plants with different Cd-accumulating abilities during the process of Cd accumulation. To test this hypothesis, a pot experiment was conducted to determine the biomass, Cd accumulation and physiological responses of the Cd hyperaccumulator S. nigrum and the non-hyperaccumulator Solanum melongena after they were exposed to 2 mg kg−1 Cd in soil and had different concentrations of IAA (0–40 mg L−1) sprayed on the surface of their leaves. Our results will provide a scientific basis for a phytohormone-based technology for the phytoremediation of metalcontaminated soil.
non-hyperaccumulator S. melongena were purchased at the Longjie Farmers’ Market of Chenggong County, Kunming City and were cultivated in clean soil in a greenhouse. After 4–5 leaves (8–10 cm height) of S. nigrum developed, seedlings of S. melongena with good growth and uniform size (with 3–4 leaves, 15–17 cm height) were selected for the pot experiment. The tested soil was collected from the pear garden of KUST and was air-dried and sieved with a 5 mm sieve. The tested soil was fully mixed according to the ratio of soil: river sand: humus = 2:1:1 and was treated with N, P, and K nutrients as the base fertilizer (N: P2O5: K2O = 0.15: 0.10: 0.15 g kg−1 soil, dry weight). The soil pH was 7.09 ± 0.02, and the contents of organic matter, total nitrogen, total phosphorus, and available potassium in the soil were 69.2 ± 1.4, 2.64 ± 0.01, 0.895 ± 0.007 and 0.22 ± 0.01 g kg−1, respectively. The concentrations of Pb, Zn and Cu in the soil were 44.39 ± 0.88, 77.35 ± 4.52 and 155.60 ± 4.29 mg kg−1, respectively, but Cd and As were below the detection limit (Cd: 0.006 mg kg−1; As: 0.011 mg kg−1). Based on the “Soil Environmental Quality: Risk Control Standard for Soil Contamination of Agricultural land” in China, the concentrations of Pb, Zn and Cu in the tested soil were all lower than the risk screening values for soil contamination of agriculture land, which are 120, 250 and 200 mg kg−1, respectively, when the soil pH ranges from 6.5 to 7.5. 2.2. Experimental design According to the results of a pre-experiment, the concentration of Cd was designed to be 2 mg kg−1, which was added in the form of CdCl2·2.5H2O. After 4 weeks of stabilization, each pot was filled with 1 kg soil. The 24 pots were randomly arranged in a randomized block design (four blocks, each block containing 3 pots for one plant) in a greenhouse. The IAA concentrations were designed to be 0, 10, 20 and 40 mg L−1, and each treatment was replicated three times. The two plant species were planted separately, and ten individuals of S. nigrum were planted in each pot. Due to the large biomass of S. melongena, four individuals were planted in each pot to ensure that the growth and biomass of the plants in each pot were similar. The plants were cultured in a greenhouse under natural light with the temperature varying from 15 to 25 °C (night/day). After 30 days, the leaf surfaces were sprayed with IAA. To guarantee the consistency of each treatment, the volume of IAA sprayed on the plant leaves in each pot was 25 mL, and three pots (replicates) were included in each IAA treatment. The control group replaced IAA with 25 mL deionized water. Due to the adaptation of the plants to IAA, they were sprayed every three days for the first two weeks and then sprayed once every seven days for the next two weeks. Spraying occurred seven times. After being sprayed with IAA the first time, the two plant species were grown for 30 days and harvested. The plant surfaces were cleaned with tap water, and the plants were divided into two parts. One part of the fresh samples was rinsed twice with deionized water, immediately frozen with liquid nitrogen, and then placed in self-sealing bags and stored at −18 °C in a refrigerator to prepare for the determination of physiological changes. The other part of the fresh samples was divided into roots, stems and leaves, and the roots of the plants were soaked in 20 mM disodium edetate (EDTA-Na2) for 15 min to remove residual Cd from the root surface. Then, the roots, stems and leaves were washed twice with deionized water. The cleaned plant materials were dried in an oven at 105 °C for 30 min and then dried at 75 °C to a constant weight for the determination of the Cd concentration.
2. Materials and methods 2.1. Plant materials and soil
2.3. Analysis methods
Seeds of the Cd hyperaccumulator S. nigrum were collected from the campus of Kunming University of Science and Technology (KUST) in July 2016. After being soaked for 10–12 h, they were disinfected with 0.2% NaClO for 10 min and were then washed with running water and deionized water several times. Then, the seeds were dried with filter paper and were spread in clean soil for germination. Seedlings of the
The soil was digested with aqua regia-HClO4, and plant samples were digested with HNO3–H2O2 for the determination of the Cd concentration. A soil sample (0.2 g) that had been passed through a 100 mesh sieve was added to 5 mL aqua regia and soaked for 12 h and was 2
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Fig. 1. Effects of IAA on biomass and height of two plants treated by 2 mg kg−1 Cd. Data are expressed as the mean value ± standard error of three replicates in each treatment (n = 3). The different letters indicate that the fresh weight and plant height have a significant difference among different concentrations of IAA treatments (P < 0.05).
heated and digested at 80 °C for 30 min, 120 °C for 60 min and 140 °C for 60 min in sequence. After grinding, the dried plant materials (0.2 g) were added to 5 mL HNO3 and heated as described above. After cooling, 1 mL of HClO4 and 1 mL 30% H2O2 were added to the soil and plant samples, which were then sequentially heated at 140 °C for 60 min. The digested samples were cooled to 25 mL, filtered, bottled and examined using an atomic absorption spectrometer (Varian AA240FS, USA). The standard materials (GBW-10048) and standard solution of Cd (GSB 041721-2004) were purchased from the National Research Centre for Certified Reference Materials. The working curve for Cd determination was y = 0.2569x+0.0021, R2 = 0.9997, where y is the absorbance value and x is the Cd concentrations (mg·L−1). The recovery of Cd was 95–98%, which met the quality control requirements for Cd determination. The root morphologies of S. nigrum and S. melongena were scanned using a scanner (HP LaserJet M 1005 MEP), and the relevant indicators, including the root length, root tip number and root surface area, were analysed using the Winrhizo root system analysis software (He et al., 2009). The root activity, which mainly reflects the absorption, synthesis, oxidation and reduction abilities of roots, was determined by the triphenyltetrazolium chloride (TTC) method (Zhang and Qu, 2003), and the ATPase activity of the plasma membrane in root cells was determined as described by Forbush (1983). Plant leaves (2.0 g) were collected and randomly mixed from each individual plant in each pot. The leaves were added to a phosphate buffer solution (PBS, pH = 7.8), homogenized in an ice bath and centrifuged at 15,000×g, 20 °C for 20 min. The supernatant was stored at 4 °C for the determination of the enzyme activities and MDA contents. The activity of superoxide dismutase (SOD) was analysed by the nitrogen blue tetrazolium method (Beyer and Fridovich, 1987), peroxidase (POD) activity was determined by the guaiacol method and catalase (CAT) activity was determined using potassium permanganate titration (Aebi, 1984; Beffa et al., 1990). The content of malondialdehyde (MDA) was determined by the thiobarbituric acid method (Heath and Packer, 1968). Additionally, the chlorophyll contents were determined by the 95% ethanol extraction method (Sartory and Grobbelaar, 1984), and the proline assay was followed by the acid ninhydrin method (Lee and Takahashi, 1966).
2.4. Statistical analysis Data were analysed by one-way and two-way analysis of variance (ANOVA) by using the statistical analysis system software (SAS 9.2), and multiple comparisons were performed using Tukey's HSD (honestly significant difference) method. The purpose of one-way ANOVA was to determine whether a significant difference existed for a given parameter among the 4 IAA treatments. There were three degree of freedom (DF) in one-way ANOVA: The DF among groups was 3 (ν1), the DF within groups was 8 (ν2) and the total DF was 11 (ν3); when ν1 = 3 and ν2 = 8, F0.05 = 4.07 and F0.01 = 7.59. Because two factors, IAA and plant species, were included in the present study, we also used two-way ANOVA to determine whether a significant difference existed for a given parameter between the plant species (Factor A), among the IAA concentrations (factor B) and for the interaction between plant species and IAA (factor A × B). There were five degree of freedom (DF) in two-way ANOVA: The DF of plants was 1 (ν1), the DF of IAA was 3 (ν2), the DF of plants × IAA was 3 (ν3), the DF of error was 16 (ν4) and the total DF was 23 (ν5). When ν1 = 1 and ν4 = 16, F0.05 = 4.49 and F0.01 = 8.53; when ν2 = ν3 = 3 and ν4 = 16, F0.05 = 3.24 and F0.01 = 5.29 (Supplementary Table 1). To compare the Cd accumulation and translocation ability between the two plants, the bioconcentration factor (BCF) and translocation factor (TF) were calculated. The BCF is defined as the ratio of the Cd concentration in shoots to that in soil, and the TF is defined as the ratio of the Cd concentration in shoots to that in roots. The level of significant difference was set at α = 0.05, and the level of extremely significant difference was set at α = 0.01. Stepwise regression analysis was performed by the statistical product and service solutions (SPSS 20.0) software, and the figures were drawn using Origin 9.0 software. 3. Results 3.1. Effect of IAA on plant growth The fresh weights of both plants were significantly higher than that of the control at the two lowest IAA treatments (10 and 20 mg L−1, P < 0.05, F = 9.575, 14.756), with maximums of 15.60 and 41.93 g 3
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Fig. 2. Effects of IAA on Cd accumulation by two plants treated by 2 mg kg−1 Cd.
plant−1, respectively, when treated with 10 mg L−1 IAA (Fig. 1a). No significant difference in the fresh weight of the two plants was observed between the 0 and 40 mg L−1 IAA treatments. The heights of both plants were significantly increased after the 20 mg L−1 IAA treatment compared to that of the control (P < 0.05, F = 10.806, 22.877, Fig. 1b), but a low concentration of IAA (10 mg L−1 IAA) significantly increased the height of S. nigrum (P < 0.05, F = 10.806).
than those in the control. However, the Cd concentrations in the shoots and roots of S. melongena were significantly increased only after the 20 mg L−1 IAA treatment compared to those in the control, and a significant decrease was observed after the highest IAA treatment (40 mg L−1, P < 0.05, F = 17.979, 12.859, Fig. 2a). In the case of BCFs, a significant increase in S. melongena was only observed after the 20 mg L−1 IAA treatment (P < 0.05, F = 17.919). For TFs, no significant changes were observed in the two plants with the addition of IAA (P > 0.05, F = 3.836, 1.173, Fig. 2c).
3.2. Effect of IAA on Cd accumulation in plants The addition of different concentrations of IAA promoted the accumulation of Cd by S. nigrum, especially after the 10 mg L−1 IAA treatment, up to 49.52 mg kg−1 in its shoots with an increasing rate of 30%. With the increase in the IAA concentration, the Cd concentrations in the roots, shoots (P < 0.05, F = 8.251, 56.994, Fig. 2a) and BCFs (P < 0.01, F = 50.254, Fig. 2b) of S. nigrum were significantly higher
3.3. Effects of IAA on the physiological and biochemical responses of plants 3.3.1. Root morphology, root activity and ATPase activity of plasma membranes IAA significantly increased the total root length, root surface area 4
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Fig. 3. Effect of IAA on root morphology of two plants treated by 2 mg kg−1 Cd.
and root tip number of S. nigrum (P < 0.05, F = 7.023–15.842, Fig. 3) at 20 mg L−1 IAA, but no significant differences were observed after the other treatments compared to those of the control. In addition, the total root length, root surface area and root tip number of S. melongena were increased significantly at the highest IAA treatment compared to those of the control (P > 0.05, F = 7.588–7.239), but no significant differences were observed between the 10 and 20 mg L−1 IAA treatments (Fig. 3). Compared to that of the control, the root activity of S. nigrum was significantly increased with the addition of IAA (P < 0.05, F = 29.394), but the root activity of S. melongena was significantly increased only after treatment with a high concentration of IAA (P < 0.05, F = 7.774). No significant difference in root activity was observed in S. melongena after the other IAA treatments (Fig. 4a). The ATPase activity of the plasma membrane from root cells of S. nigrum and S. melongena were significantly increased when treated with low and high concretions of IAA (P < 0.05, F = 22.537, 8.932, Fig. 4b), but no significant difference was observed for the other IAA treatments compared to the control.
3.3.2. Effect of IAA on the activities of antioxidant enzymes and the proline and MDA contents of plants The activities of antioxidant enzymes in the leaves of plants varied with the addition of different concentrations of IAA. The SOD activity in the leaves of S. nigrum was significantly increased compared to that in the control when treated with 10 mg L−1 IAA (P < 0.05, F = 134.023, Fig. 5a), but the SOD and CAT activities in this plant were significantly decreased when treated with a high concentration of IAA (P < 0.05, F = 134.023, 51.474, Fig. 5a, c). In the case of S. melongena, the POD and CAT activities were also significantly increased after treatment with a high concentration of IAA (P < 0.05, F = 15.31, 42.551), but a significant increase in SOD activity was only observed after the 20 mg L−1 IAA treatment (P < 0.05, F = 40.342, Fig. 5). With the increasing concentrations of IAA, the content of malondialdehyde (MDA) in leaves of the plants was significantly decreased (P < 0.05, F = 9.583, 20.626, Fig. 5d). When leaves were sprayed with 20 mg L−1 IAA, the MDA content in the leaves of S. nigrum reached the minimum value (0.015 μmol g −1). For S. melongena, the MDA content in the leaves of this plant was significantly decreased compared to that in the control, except after the 10 mg L−1 IAA treatment 5
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Fig. 4. Effect of IAA on root activity and ATPase activity of plasma membrane in root cells treated by 2 mg kg−1 Cd.
(Fig. 5d). The proline contents of the plants were significantly increased after the two lowest IAA treatments compared to that in the control (P < 0.05, F = 24.778, 17.7, Fig. 5e). However, the proline contents of the two plants were not significantly different from that of the control when sprayed with a high concentration of IAA (P > 0.05, Fig. 5e).
significant negative correlation with CAT (X9) and chlorophyll a (X11). The regression equation for S. melongena was Y = 6.391 + 1.75 E005X7-0.349X9-1.959X11 (P < 0.01, r = 0.964, F = 35.188).
3.3.3. Effect of IAA on the contents of photosynthetic pigments As shown in Table 1, the contents of photosynthetic pigments (chlorophyll a, b and carotenoids) in the two plants were significantly higher than those of the control (P < 0.05, F = 4.799, 14.778) when treated with 10 mg L−1 IAA. There was no significant difference among the other IAA treatments compared to the control. The results of twoway analysis of variance (ANOVA) showed that the contents of photosynthetic pigments in plants were significantly affected by the concentration of IAA (P < 0.01, Table 2). The results of two-way ANOVA showed that plant height, Cd accumulation, root surface area, root tip number, root activity, ATPase activity of plasma membrane in root cells, SOD and POD activities, and MDA content were significantly affected by the plant species, IAA concentration and their interactions (P < 0.01, Figs. 1–5).
Cadmium is toxic to plant growth, development and reproduction. Plant growth is slower when the Cd concentration in the growth medium increases. When present in soil at a high concentration, Cd destroys the chloroplast structure of plants, leading to yellowing or necrosis (Shi et al., 2010). The Cd concentration used in this study was 2 mg kg−1 according to our previous experiment because no visible toxicity symptoms were observed in two tested plants at this Cd concentration. It was also reported that after treatment with 2 mg kg−1 Cd, the biomasses of the aboveground and underground parts of S. nigrum were not significantly different from those of the control (Gao et al., 2010; Jiao et al., 2013). Moreover, it was found that when the Cd concentration of soil was 2 mg kg−1, the 13 varieties of S. melongena studied essentially grew normally and showed no symptoms, such as chlorosis or green deficiency, indicating that the two plants could grew well and had high tolerance to Cd at this Cd concentration. However, the Cd tolerance of S. nigrum was stronger than that of S. melongena. Wei et al. (2005) planted S. nigrum in soil containing 0, 10, 25, 50, 100 and 200 mg kg−1 Cd and found that neither the average height nor the shoot dry mass of S. nigrum was reduced significantly after the 10 and 25 mg kg−1 Cd treatments, respectively. Nevertheless, the average height of S. nigrum was significantly reduced (P < 0.05) after the 50, 100 and 200 mg kg−1 Cd treatments. Comparatively, the tolerance of S. melongena for Cd was weak and its growth was significantly reduced by 3 mg kg−1 Cd (Singh and Prasad, 2014). As a plant growth regulator, IAA is widely used to increase the growth of plants (Zhu et al., 2013). Khan et al. (2019) reported that compared to Cd treatment alone, the fresh weights of root and shoots in tomato seedlings were significantly increased by 16.3% and 11.17% after adding 5 μM IAA under 100 μM Cd stress, respectively. In the present study, application of the two lowest IAA concentrations was associated with a significant increase in fresh weight of both plants, and the shoot Cd concentration in S. nigrum increased (Fig. 1a). This result indicated that IAA could promote the growth of the hyperaccumulator
4. Discussion 4.1. Plant growth and tolerance to Cd are improved by IAA
3.4. Stepwise regression analysis A multiple stepwise regression analysis was performed to test the correlation among the different indexes. Here, Y is the Cd concentration in the leaves of plant, X1 is the IAA treatment concentration, X2 is the total root length, X3 is the root surface area, X4 is the root tip number, X5 is the root activity, X6 is the ATPase activity of the plasma membrane in root cells, X7 is the SOD activity, X8 is the POD activity, X9 is the CAT activity, X10 is the MDA content, X11 is chlorophyll a, X12 is chlorophyll b, X13 is carotenoid and X14 is the proline content. The regression equation for S. nigrum was Y = 11.705–12.369X11+86.191X13+1.059X14 (P < 0.01, r = 0.947, F = 23.008). Therefore, the Cd concentration in leaves of S. nigrum was significantly and positively correlated with carotenoids (X13) and the proline content (X14) and negatively correlated with chlorophyll a (X11). There was a significant positive correlation between the Cd concentration and SOD activity (X7) in leaves of S. melongena and a 6
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Fig. 5. Effect of IAA on activities of antioxidative enzymes, malondialdehyde (MDA) and proline contents in plants treated by 2 mg kg−1 Cd.
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Table 1 Effect of IAA on the photosynthetic pigment contents of plants at 2 mg kg−1 Cd treatment. Plant species
IAA concentration (mg L−1)
S. nigrum
0 10 20 40 0 10 20 40
S.melongena
Photosynthetic pigment contents (mg·g−1) Chlorophyll a 1.03 ± 0.13 b 1.43 ± 0.05a 1.11 ± 0.14 ab 1.15 ± 0.19 ab 1.11 ± 0.02 b 1.22 ± 0.03a 1.14 ± 0.02 b 1.21 ± 0.02a
Chlorophyll b 0.46 ± 0.05 b 0.71 ± 0.08a 0.55 ± 0.08 ab 0.51 ± 0.09 ab 0.45 ± 0.02 b 0.56 ± 0.04a 0.51 ± 0.02 ab 0.52 ± 0.02 ab
Carotenoid 0.22 ± 0.03 b 0.30 ± 0.02a 0.23 ± 0.03 b 0.26 ± 0.01 ab 0.24 ± 0.01 b 0.29 ± 0.03a 0.25 ± 0.01 ab 0.28 ± 0.02a
Total photosynthetic pigments 1.49 ± 0.18 b 2.15 ± 0.22a 1.66 ± 0.22 ab 1.67 ± 0.28 ab 1.56 ± 0.08 b 1.78 ± 0.07a 1.66 ± 0.08 ab 1.72 ± 0.06 ab
Data are expressed as the mean value ± standard error of three replicates in each treatment (n = 3). The different letters in the column of each index indicate that there is a significant difference among different concentrations of IAA treatments in a plant species (P < 0.05).
S. nigrum and enhance its tolerance to Cd. On the other hand, the heights of the two plants varied after administration of IAA (Fig. 1b), possibly due to the difference in plant species, which makes plant tissue sensitive to IAA. Ji et al. (2015) found that IAA can effectively increase the biomass and Cd concentration of shoots in S. nigrum. In our study, the shoot Cd concentration in S. nigrum was significantly improved after IAA application. However, this obvious shoot Cd enhancement was only observed after the 20 mg L−1 IAA treatment in S. melongena (Fig. 2a). This result indicates that the Cd accumulation in shoots of S. nigrum is enhanced by a low concentration of IAA, although its TF values showed no significant change (Fig. 2c). Compared to S. nigrum, the accumulation of Cd in S. melongena was enhanced by IAA at a higher level.
was also markedly increased (Fig. 3), indicating that IAA could improve the root morphology of the Cd hyperaccumulator by promoting the division and differentiation of plant cells, which may be beneficial to Cd accumulation. Additionally, the root morphology of S. melongena was also significantly improved after the highest IAA treatment (Fig. 3). Root activity is a physiological indicator that can be used to characterize plant root growth and stress resistance. In this study, the root development of S. nigrum was notably promoted by application of IAA. Accordingly, the root activity of S. nigrum was improved by application of IAA, but an obvious increase in root activity in S. melongena was only shown after the highest IAA treatment (Fig. 4a). This result indicated that the root activity of S. nigrum was facilitated by low concentrations of IAA, which in turn, contributed to plant growth. Additionally, the ATPase activity of the plasma membrane in root cells in S. nigrum and S. melongena was obviously increased when treated with 10 and 40 mg L−1 IAA, respectively (Fig. 4b). It has been documented that exogenous application of IAA significantly increases H+-ATPase activity in the plasma membrane in Al-stressed alfalfa roots and promotes H+ secretion from root tips (Wang et al., 2017). The decreasing pH value in the rhizospheric environment changes the bioavailability and mobilization of Cd and affects Cd uptake by plants.
4.2. IAA improves the root morphology of plants Generally, the root system is the first organ to respond to stress, and plants adapt to environmental stress by changing their root morphology and distribution (Takahashi and Asada, 1983). Qin et al. (2018) reported that the root length, root volume, root dry matter weight, root effective surface area and total root area of winter wheat decreased with an increasing Cd concentration. However, IAA treatment promotes plant cell division, root growth and root surface areas. The root growth of sunflowers was effectively promoted by 3 or 6 mg L−1 IAA (Liphadzi et al., 2006). Our results showed that the total root length, root surface areas and root tip numbers of S. nigrum were obviously increased with the addition of 20 mg L−1 IAA, and the Cd concentration in S. nigrum
4.3. IAA promotes Cd accumulation in the two plants by different physiological mechanisms Superoxide dismutase (SOD) is an important protective enzyme in plants. Under normal conditions, the activity of SOD in plants is not high,
Table 2 Two-way ANOVA for photosynthetic pigment contents. Index
Source
Degree of freedom
Sum of squares
Mean square
F ratio
F0.05
F0.01
P value
Chlorophyll a
Plant IAA Plant Error Total Plant IAA Plant Error Total Plant IAA Plant Error Total Plant IAA Plant Error Total
1 3 3 16 23 1 3 3 16 23 1 3 3 16 23 1 3 3 16 23
0.001 0.225 0.081 0.16
0.001 0.075 0.027 0.01
0.093 7.439** 2.729
4.49 3.24 3.24
8.53 5.29 5.29
0.764 0.002** 0.078
0.013 0.099 0.021 0.048
0.013 0.033 0.007 0.003
3.753 9.947** 2.165
4.49 3.24 3.24
8.53 5.29 5.29
0.071 0.001** 0.132
0.001 0.018 0.003 0.0096
0.001 0.006 0.001 0.0005
1.657 13.212** 1.212
4.49 3.24 3.24
8.53 5.29 5.29
0.216 0.000** 0.337
0.023 0.618 0.183 0.448
0.023 0.206 0.061 0.028
0.807 7.288** 2.148
4.49 3.24 3.24
8.53 5.29 5.29
0.382 0.003** 0.134
Chlorophyll b
Carotenoid
Total photosynthetic pigments
× IAA
× IAA
× IAA
× IAA
The double asterisk (**) shows the difference is very significant (P < 0.01). 8
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but it is increased to eliminate peroxides in plants under adverse conditions, thus maintaining the normal metabolism of plants (Tian et al., 2012). Peroxidase protects cells from damage by removing excess lipid peroxidation products from the body (Doğanlar, 2013). Catalase is a catalyst that hydrolyses H2O2 into H2O and O2, thereby avoiding the risk of peroxidation in membrane lipids. Exogenous IAA can resist metal stress by regulating the activities of antioxidant enzymes (SOD, POD and CAT etc.) in plants (Bashri and Prasad, 2016). In this study, the activities of POD and CAT in S. nigrum and the activity of SOD in S. melongena were notably increased by 20 mg L−1 IAA (Fig. 5), indicating that the ability of S. nigrum to remove reactive oxygen species (ROS) was stronger than that of S. melongena after the addition of IAA. The stepwise regression analysis results showed that the Cd concentration in leaves of S. melongena was significantly and positively correlated with SOD activity and negatively correlated with CAT activity. Malondialdehyde is a peroxidative product of membrane lipids in plant cells, and its content reflects the level of membranous peroxidation in plants to some extent. The level of MDA in leaves of T. foenum-graecum was significantly reduced after a 10 μM IAA treatment under different concentrations of Cd stress (Bashri and Prasad, 2016). Our study found that the MDA content in both plants obviously decreased after IAA application, but after the treatment with 20 mg L−1 IAA, the MDA content in leaves of S. nigrum was much lower than that in S. melongena (Fig. 5d). This result indicates that the addition of IAA reduced the peroxidation degree of membrane lipids in the two plants and increased their tolerance to Cd. However, compared to S. melongena, S. nigrum had a more effective defence system to decrease the peroxidation of membrane lipids. The contents of chlorophyll in plants can be used as an indicator to measure the photosynthetic capacity and the damage degree of plants in response to stress. It has been reported that the application of IAA to Chlorella vulgaris has a beneficial effect on the photosynthetic apparatus under metal stress (Piotrowska-Niczyporuk et al., 2012). In the present study, the contents of photosynthetic pigments in the leaves of the two plants were markedly increased when treated with 10 mg L−1 IAA (Table 1). This result indicated that treatment with a suitable concentration of IAA could promote photosynthesis in S. nigrum and S. melongena, which is beneficial to the accumulation of photosynthetic pigments in the leaves of plants, but this promotion effect on S. nigrum was more obvious (Table 1). Stepwise regression analysis showed that the Cd concentration in the leaves of the plants was significantly and negatively correlated with the chlorophyll a content, probably due to the inhibition of chlorophyll biosynthesis by Cd (Singh and Prasad, 2015) and because chlorophyll a is more sensitive than other photosynthetic pigments. Additionally, the Cd concentration in the leaves of S. nigrum was significantly and positively correlated with carotenoids, probably because carotenoids can protect photosynthesis under stress by removing singlet oxygen (Singh and Prasad, 2015), thereby increasing plant biomass and promoting Cd accumulation by S. nigrum. Additionally, free proline is an osmotic adjustment substance in plants, and its content can reflect the stress resistance of plants to some extent. Proline can form a nontoxic proline-metal complex by chelation, thereby enhancing plant tolerance to stress (Sun et al., 2007). In the present study, the proline content of the plants was obviously increased after the lowest two IAA treatments, but the increase in S. nigrum was higher than that in S. melongena (Fig. 5e). The stepwise regression analysis results also showed that the Cd concentration in leaves of S. nigrum was significantly and positively correlated with the proline content. Therefore, IAA can enhance the resistance of S. nigrum by adjusting its proline level. On the whole, exogenous addition of IAA could promote the accumulation of Cd in leaves by promoting physiological indicators, which were different in the Cd hyperaccumulator and non-hyperaccumulator.
synthetic PGRs are stable and cheap. Therefore, PGRs are widely applied in agricultural production. During our experimental design, we did not realize the difference between native IAA and synthetic Dicamba or NAA (naphthalene acetic acid), and native IAA is popularly used in some references (Fässler et al., 2010; Agami and Mohamed, 2013; Singh and Prasad, 2015). We will consider the difference of these two phytohormones from different sources in the future, and a comparative experiment is needed to determine the effects of native IAA and synthetic Dicamba/NAA at the same concentration on plant Cd uptake and physiological responses. Regarding the efficiency of IAA application under field conditions, little information is available on the phytoremediation efficiency of metals in soil because many studies have focused on pot experiments, such as those of Liphadzi et al. (2006), Ji et al. (2015b) and Bashri and Prasad (2016). However, IAA is successfully used in the field to alleviate salinity stress in maize (Kaya et al., 2013) and improve the net photosynthetic rate of Zizania latifolia (Li et al., 2019). At present, we are conducting a field experiment in Cd–Pb–As combined polluted soil in the suburb of Gejiu City, Yunnan Province to examine the Cd phytoextraction efficiency of Solanum nigrum by IAA or/and kinetin application. 5. Conclusions Our work indicated that under the stress of 2 mg kg−1 Cd, the growth of S. nigrum and S. melongena was significantly increased by the addition of 20 mg L−1 IAA, which also improved the osmotic adjustment and activities of their antioxidant enzymes and alleviated the lipid peroxidation of cell membranes. The shoot Cd concentration in S. nigrum was significantly increased with the application of 10–40 mg L−1 IAA, but this significant increase of shoot Cd was only observed at 20 mg L−1 IAA in S. melongena. Furthermore, the Cd concentration in shoots of S. nigrum was significantly and positively correlated with the contents of carotenoid and proline, and there was a significant positive correlation between the Cd concentration and SOD activity in leaves of S. melongena. Therefore, exogenous addition of IAA could enhance the accumulation of Cd in shoots by promoting physiological indicators, which were different in the Cd-hyperaccumulator and non-hyperaccumulator. It should also be noted that with the addition of IAA, S. nigrum showed a stronger ability to accumulate Cd in shoots compared to that in S. melongena. Compared to chelating agent-induced phytoextraction technology, native phytohormones or other PGRs are widely used in agricultural production. IAA is easily absorbed by plants and is degraded in plants by enzymatic reactions. Therefore, a significant negative effect on soil quality may be avoided if a suitable concentration of IAA is added. However, our present work was conducted in a greenhouse with a pot experiment, which cannot fully reflect the actual situation in the field. Therefore, this IAA-assisted Cd phytoextraction should be further tested in the field during the phytoremediation of Cdcontaminated soil. CRediT authorship contribution statement Jiakang Ran: Investigation, Writing - original draft, Writing - review & editing. Wen Zheng: Investigation, Data curation, Formal analysis. Hongbin Wang: Conceptualization, Methodology, Writing original draft, Writing - review & editing. Haijuan Wang: Conceptualization, Methodology, Writing - review & editing. Qinchun Li: Formal analysis, Writing - review & editing.
4.4. Potential of IAA-induced phytoextraction technology in practice
Acknowledgments
It should be noted that we used native auxin in our experiment, and it is easily degraded in the natural environment. Comparatively,
This work was supported by the Key Technologies Research and Demonstration Project for Yunnan's Soil Remediation (2018BC004–2). 9
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Appendix A. Supplementary data
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