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The physiological response of Mirabilis jalapa Linn. to lead stress and accumulation
International Biodeterioration & Biodegradation xxx (2016) 1e4
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
The physiological response of Mirabilis jalapa Linn. to lead stress and accumulation Jun Wang a, Sheng Ye b, Shengguo Xue a, *, William Hartley c, Hao Wu a, Lizheng Shi a a
School of Metallurgy and Environment, Central South University, Changsha 410083, PR China Jiangsu Sentay Environmental Science and Technology Co., Ltd., Nanjing 211153, PR China c Crop and Environment Sciences Department, Harper Adams University, Newport, Shropshire, TF10 8NB, United Kingdom b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2016 Received in revised form 13 April 2016 Accepted 23 April 2016 Available online xxx
Smelters, metal refineries and mining operations have all been considered as major sources of metal release into the environment. As a highly toxic and cumulative poison, once in the environment Pb is difficult to remove and can adversely affect human health. Mirabilis jalapa Linn. (The Marvel of Peru) is a fast growing plant that shows potential for phytostabilization of Pb contaminated soils. Fourier Transform Infrared (FTIR) spectrometry was adopted to detect physiological changes in the chemical composition of M. jalapa exposed to six different concentrations of Pb in solution (0, 50, 100, 200, 500 and 1000 mmol L1 Pb). Results indicated that biomass was reduced in plants grown in Pb treatments compared to controls, although M. jalapa grew typically well at the greatest concentration, 1000 mmol L1. The concentration of Pb in plant tissues occurred in the order roots > leaves > stems, with a translocation factor of less than 0.04. The absorbance of dominating bands near 3420, 2920, 1610 and 1060 cm1 firstly increased but then declined in root tissues; the bands respectively corresponding to organic acids, carbohydrate, protein and amino acids. However, no obvious changes were observed in leaves and stems. The results suggest that M. jalapa can reduce transportation of Pb from roots to shoots, subsequently preventing Pb toxicity in shoots. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Mirabilis jalapa Linn. Pb toxicity FTIR Chemical composition Phytostabilization
1. Introduction Lead (Pb) is one of the main heavy metal pollutants in the environment. With the development of modern industry, Pb presents a marked increase in the environment due to mining growth, over-application of chemical fertilizers and vehicle exhaust emissions (Pietrzykowski et al., 2014). As a non-essential element for plants, Pb induces metabolic disorders, inhibits growth and development, and following excessive exposure, even death. Furthermore, Pb threatens human health through food chain transfer. Therefore, treatment of soil contaminated by Pb has become a priority (Li et al., 2013). Smelters, metal refineries and mining operations have all been considered as major sources of heavy metal release into the environment (Yu and Gu, 2008). Mine tailings in particular are difficult to manage due to heavy metal contamination, which makes plant establishment difficult as a result of poor physical soil structure and
* Corresponding author. E-mail addresses: [email protected], [email protected] (S. Xue).
elevated metal toxicity (Xue et al., 2016a; Zhu et al., 2016). Establishment of plant tolerance can reduce heavy metal bioavailability (Van der Ent A et al., 2012; Xue et al., 2016b). Plants can be classified into three main groups according to metal uptake characteristics: (i) Excluders, restrict translocation. (ii) Index plants, uptake and translocation reflect soil metal concentrations. (iii) Accumulators, plants actively concentrate metals in their tissues. There is evidence to suggest that higher plants have mechanisms to protect themselves from metal toxicity. These mechanisms include metal sequestration using organic compounds (metal-binding polypeptides), subcellular compartmentalization (generally removal of metals to the cell vacuole), active metal efflux (excreting metals by active pumps) and organic ligand exudation (organic molecules exuded by root cells) (Komal et al., 2015). Two mechanisms for heavy metal tolerance in plants have been proposed. The first is exclusion, meaning that heavy metals are absorbed by plants but then subsequently detached through active transport or aging of organs in vitro to make them discharge (Kiran and Thanasekaran, 2011). The second mechanism is cumulative, whereby the heavy metal becomes non-bioactive following detoxification. Examples include Pb precipitation on cell walls of
http://dx.doi.org/10.1016/j.ibiod.2016.04.030 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang, J., et al., The physiological response of Mirabilis jalapa Linn. to lead stress and accumulation, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.04.030
2
J. Wang et al. / International Biodeterioration & Biodegradation xxx (2016) 1e4
rice which prevent excessive Pb from being transported into the protoplast (Wu et al., 2016), and removal of Cr from hydroponic solution in hybrid willows without showing detectable phytotoxicity (Yu and Gu, 2007). Lead can also be transferred to the vacuole, so as to reduce its concentration in protoplasts. Organic acids and proteins can precipitate Pb by chelation thereby reducing its free state. Mirabilis jalapa (The Marvel of Peru) demonstrates tolerance, rapid growth, and characteristics of native plants found in China, which is an ideal germplasm resource to restore soils contaminated by heavy metals (Zhou et al., 2012). When plants are stressed by heavy metal contamination a change in growth, development and characteristics of accumulation occurs (Liang et al., 2011; Xu et al., 2012). In this paper, the enrichment concentration and tolerance of lead were investigated on hydroponically grown M. jalapa. Fourier transform infrared spectroscopy was used to determine chemical changes in different tissues and organs of M. jalapa when it was stressed by lead uptake (Bosch et al., 2006; Gu et al., 2009; Ohno et al., 2015). The objectives of the present study were to interpret M. jalapa's physiological response to Pb toxicity and determine if it could tolerate high concentrations of Pb. 2. Materials and methods 2.1. Experiment method M. jalapa seeds, collected from the tailings wasteland at the Xiangtan manganese mine, were sprinkled on sand-filled pots. Following germination (14 days), M. jalapa seedlings of the same size were selected, the roots were gently washed with tap-water and then thoroughly rinsed with deionized water. Plants were then grown in black plastic containers supplied with 20 L of Hoagland nutrient solution and exposed to 6 concentrations of Pb: 0 (control), 50, 100, 200, 500 and 1000 mmol L1, added as Pb(NO3)2(AR). Treatments in a glass house were replicated three times. All solutions were continuously aerated and pH maintained at 4.5 using 0.1 M NaOH or 0.1 M HCl, to ensure that Pb remained stable as Pb2þ in an ionic state. Plants were harvested after 35 days growth. Plant roots were then gently washed with tap water and then subsequently washed with deionized water and finally blotted dry with tissue paper. Plant samples were divided into roots, stems and leaves, which were firstly oven dried at 105 C for 30 min and then at 75 C until a constant weight was achieved (48 h). Fresh weight (hereafter referred to as FW) and dry weight (hereafter referred to as DW) of roots, stems and leaves were recorded. Samples of dry plant material for Pb analysis were ground to a fine powder using a stainless steel grinder, so as to pass through a 200 screen mesh.
using an agate pestle and mortar. Each sample was thoroughly mixed with sodium bromide particles and tableted into three almost transparent wafers. The samples were scanned 32 times and the three replicate spectra averaged to account for within sample variability and differences in particle size and packing density. 2.4. Statistical analysis All analyses were performed in quintuplicate. The data were statistically treated with Microsoft Excel 2003, SPSS version 19.0 and Origin 8.0. In the case of homogeneity, Duncans post hoc test was used. If there was no homogeneity, Dunnetts T3 test was performed. All figures were constructed using Origin 8.0. 3. Results and discussion 3.1. Growth response of M. jalapa to Pb concentrations M. jalapa grew normally under the range of Pb concentrations. However, at high concentrations (1000 mmol L1), the leaves rolled up slightly and the roots became black. Nevertheless, the different concentrations of Pb did not affect biomass production (Fig. 1). Therefore, a number of concentrations of Pb played a small role in M. jalapa growth and M. jalapa has a strong Pb tolerance ability in the growth medium. 3.2. Pb uptake and accumulation characteristics of M. jalapa With an increase in Pb concentration, Pb accumulation increased in all parts of the plant (Table 1). The content of Pb was found in the order roots > leaves > stems. Even with different Pb concentrations, the content of Pb in stems was the same as that in the control (P > 0.05). However, Pb increased significantly when plants were exposed to high concentrations of 500 and 1000 mmol L1 (P < 0.05) (see Table 2). Translocation factor (Hereafter referred to as TF) reflects the transportation and distribution of metals in plants from below ground to above. A number of studies (Du et al., 2013; Tabaraki et al., 2014; Yin et al., 2011) have shown that Pb mostly accumulates in the roots, which therefore reduces transportation to the above ground tissues, which is one of the important mechanisms to reduce toxicity and has been demonstrated in this study. No clear trend was observed in the TF of M. jalapa stem and leaf tissues.
2.2. Pb content analysis Subsamples of dried plant tissue (c. 0.15 g) were digested with 90 mL of concentrated HNO3 (AR, mass fraction ¼ 65%), 30 mL of HCl (AR, mass fraction ¼ 36e38%) and 3 mL of HClO4 (AR, mass fraction ¼ 70e72%) in a block heater. Lead was determined using ICP-OES (Jones et al., 2011). 2.3. FTIR analysis The spectral information of various tissues and organs was characterized using Fourier transform infrared (FTIR) spectroscopy in the mid-IR range with a Nicolet IS10 infrared spectrometer. The detector was deuterated triglycine sulphate (DTGS). The measured wavebands ranged from 4000 to 500 cm1 with a resolution of 1 cm1. Plant samples were finely ground with KBr (0.5/50 mg)
Fig. 1. Effects of various concentrations of Pb on the biomass of M. jalapa.
Please cite this article in press as: Wang, J., et al., The physiological response of Mirabilis jalapa Linn. to lead stress and accumulation, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.04.030
J. Wang et al. / International Biodeterioration & Biodegradation xxx (2016) 1e4
3.3. FTIR analysis of M. jalapa
Table 1 Effect of various concentrations of Pb on its accumulation in M. jalapa. Concentration/mmol$L1 Pb accumulation/mg kg1
0(CK) 50 100 200 500 1000 Significance level
3
Root
Stem
Leaf
22.9 ± 17.0a 3292.1 ± 959.3b 3968.9 ± 536.3bc 5228.5 ± 1294.1bc 6725.2 ± 128.7bc 9313.3 ± 4344.2c <0.05
3.6 ± 1.1a 45.2 ± 5.1a 74.3 ± 27.7ab 94.2 ± 26.7abc 123.6 ± 103.8bc 247.7 ± 198.3c <0.05
4.4 ± 1.5a 58.6 ± 2.9a 138.4 ± 49.4b 143.6 ± 29.5b 212.6 ± 13.2c 263.1 ± 68.5c <0.05
Table 2 Translocation factor of Pb in M. jalapa at various concentrations of Pb. Concentration/mmol L1
TF in stem
TF in leaf
50 100 200 500 1000
0.014 0.019 0.018 0.018 0.027
0.018 0.035 0.027 0.032 0.028
TF: Translocation factor.
However, the TF was less than 0.04, as Pb is difficult to be transport to above ground tissues. The possible reason is Pb adsorption, sedimentation and passivation, which then accumulated in the roots of M. jalapa. In addition, this demonstrates that M. jalapa has a very strong Pb tolerance. At the same time, TF of Pb accumulation in the leaves is higher than that in the stems with various concentrations of Pb.
In Fig. 2A, the stretching vibration peak of 3420 cm1 (free hydroxyl) is mainly reflected in root carbohydrate (cellulose, hemicelluloses, polysaccharides) (Ren et al., 2008). The peak of absorbance increases with the highest peak at 200 mmol L1, and then the absorbance of the peak reduces with an increase in the concentration of Pb. This suggests that low concentrations of Pb promote the secretion of carbohydrates in M. jalapa roots, while high concentrations of Pb inhibit the secretion of carbohydrates. As Pb concentrations continued to increase, root epidermal cell walls absorb and combine with Pb2þ which then subsequently weakens the hydrogen bonds of the cell wall surfaces. The carboxylic acid OeH and methyl stretching vibration peaks overlap near 2920 cm1, which is mainly from various vitamins, membrane and cell wall components. Absorbance increased in the medium and low Pb concentrations, and then decreased with an increase in Pb concentrations (Fig. 2A). It may be that in the medium and low concentrations, M. jalapa roots continuously secrete chelating organic acids as a mechanism of resistance increasing the carboxylic acid bands. Increasing Pb concentrations may lead to physiological and biochemical imbalances, which may have had an adverse effect on the plants resistance mechanisms and resulted in reduced secretion of organic acids. The bending vibration peak of NeH amide near 1655 cm1e1590 cm1 is characteristic of protein (Ren et al., 2008). Amino acid, peptide and protein substances are also root exudates produced in response to Pb toxicity (Li et al., 2013). In medium and low Pb concentrations, Pb stimulated the synthesis and secretion of these substances to complex Pb and hence reduce Pb toxicity to the plants. However, increased concentrations of Pb may have inhibited the synthesis of these substances. Fig. 2A reveals that the peak near 1060 cm1 is a stretching vibration peak of alcohol and ether-based ester or phenol group CeO
Fig. 2. Absorption FTIR spectra in M. jalapa. A: in roots of M. jalapa; B: in stems of M. jalapa; C: in leaves of M. jalapa. a: 0(CK); b: 50 mmol L1; c: 100 mmol L1; d: 200 mmol L1; e: 500 mmol L1; f:1000 mmol L1.
Please cite this article in press as: Wang, J., et al., The physiological response of Mirabilis jalapa Linn. to lead stress and accumulation, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.04.030
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J. Wang et al. / International Biodeterioration & Biodegradation xxx (2016) 1e4
bond. With lower Pb concentrations, a possible reason for the increase in this peak may be synthesis of these compounds by the plant production of hydroxyls that chemically absorbed Pb2þ. However, when Pb continued to increase, transportation of these compounds to the roots was affected and peak height therefore reduced. Fig. 2B displays the IR spectra of M. jalapa stems. The peak near 1060 cm1 is a stretching vibration peak of alcohol and ether-based, ester or phenol groups. The peak near 1630 cm1 is amino acid, peptide and protein substances of bending vibration amide NH peaks. The peak near 2920 cm1 is a carboxylic acid-type OeH stretching vibration peak of organic acids substances. The peak near 3420 cm1 are carbohydrates (cellulose, hemicelluloses, polysaccharides substances). Fig. 2B reveals that no obvious changes were observed in the remaining peaks except that irregular changes occurred in the 3420 cm1 peak. As the Pb concentration increased, organic components did not change significantly and therefore at a certain range of external Pb concentration, physiological and biochemical processes were less affected, which indicates that M. jalapa possesses strong Pb tolerance. Lead concentration in M. jalapa stems did not change significantly with the increase in Pb concentration. Additionally, this shows that Pb in M. jalapa stem had been impeded. Fig. 2C displays IR spectra from M. jalapa leaves grown in different Pb concentrations. The peak near 3420 cm1 is an intermolecular hydrogen bond OeH stretching vibration of free hydroxyls. At 2920 cm1 a carboxylic acid OeH stretching vibration peak overlapping with a methyl stretching vibration is revealed. The peak near 1650 cm1 is an amide NeH bending vibration peak and near 1380 cm1 are oils and fats containing compounds of methyl CeH deformation vibration peaks. The peak near 1060 cm1 is alcohol- and ether-based, ester or a phenol group of the CO stretching vibration peak. Based on Fig. 2C, the peak heights indicate tolerance characteristics of M. jalapa to Pb. The physiological and biochemical processes of M. jalapa leaves appear not to have been affected have not been significantly affected. There are some reports indicating that heavy metals can induce protein synthesis, such as proline-rich protein, disease-related protein and histidine-rich protein (Gu et al., 2009). Besides it is believed that these heavy metalinduced proteins may have the capability to protect plant cells from heavy metal poisoning (Bosch et al., 2006). The bending vibration peak height of NeH amide near 1650 cm1 was higher when the Pb concentrations increased, which may be related to increased amino acid, peptides and protein production in M. jalapa leaves. 4. Conclusions M. jalapa is resilient to Pb, and was not affected when the concentration of Pb reached 1000 mmol L1. Lead accumulated in the roots, thereby reducing its toxicity to the aerial parts of the plant. FTIR studies demonstrated that there were fluctuations in the production of carboxylic acids, carbohydrates, proteins and amino acids in its roots. These increased with low concentrations of Pb but decreased with higher concentrations. The absorbance of dominating bands near 3420, 2920, 1610 and 1060 cm1 firstly increased but then declined in root tissues; these bands respectively corresponding to organic acids, carbohydrate, protein and amino acids. However, no obvious changes were observed in leaves and stems. Due to M. jalapa's large biomass and resistance to Pb toxicity, it
shows future promise for rehabilitating mine tailings highly contaminated with Pb. Acknowledgements Financial support from National Natural Science Foundation of China (No. 40771181) and Environmental protection's special scientific research for Chinese public welfare industry (No. 201109056) is gratefully acknowledged. References Bosch, A., Serra, D., Prieto, C., 2006. Characterization of Bordetella pertussis growing as biofilm by chemical analysis and FTIR spectroscopy. Appl. Microbiol. Biotechnol. 71, 736e747. Du, L.N., Zhao, M., Li, G., Xu, F.C., Chen, W.H., Zhao, Y.H., 2013. Biodegradation of malachite green by Micrococcus sp. strain BD15: biodegradation pathway and enzyme analysis. Int. Biodeterior. Biodegrad. 78, 108e116. Gu, Y.H., Liu, P., Chai, Q.M., Chen, J., Xie, H.K., 2009. Physiological characteristics of FTIR combined results of cadmium stress on the impact of gray moss. Spectrosc. Spectr. Analysis 29, 620e623. Jones, B.E.H., Haynes, R.J., Phillips, I.R., 2011. Influence of organic waste and residue mud additions on chemical, physical and microbial properties of bauxite residue sand. Environ. Sci. Pollut. Res. 18, 199e211. Kiran, B., Thanasekaran, K., 2011. Metal tolerance of an indigenous cyanobacterial strain, Lyngbya putealis. Int. Biodeterior. Biodegrad. 65, 1128e1132. Komal, T., Mustafa, M., Ali, Z., Kazi, A.G., 2015. Heavy metal uptake and transport in plants. Heavy Metal Contam. Soils 44, 181e194. Li, M., Cheng, X.H., Guo, H.X., 2013. Heavy metal removal by biomineralization of urease producing bacteria isolated from soil. Int. Biodeterior. Biodegrad. 76, 81e85. Liang, W.B., Xue, S.G., Shen, J.H., Wang, P., Wang, J., 2011. Manganese stress on morphological structures of leaf and ultrastructures of chloroplast of a manganese Hyperaccumulator-Phytolacca americana L. Acta Ecol. Sin. 31, 1e7. Ohno, K.M., Clausen, C.A., Iii, F.G., Diehl, S.V., 2015. Insights into the mechanism of copper-tolerance in Fibroporia radiculosa: the biosynthesis of oxalate. Int. Biodeterior. Biodegrad. 105, 90e96. Pietrzykowski, M., Socha, J., Doorn, N.S.V., 2014. Linking heavy metal bioavailability (Cd, Cu, Zn and Pb) in Scots pine needles to soil properties in reclaimed mine areas. Sci. Total Environ. 470, 501e510. Ren, L., Cheng, Z., Liu, P., Li, Z., 2008. Studies on the physiological response of Phytolacca americana to manganese toxicity by FTIR spectroscopy. Spectrosc. Spectr. Analysis 28, 582e585. Tabaraki, R., Nateghi, A., Ahmady-Asbchin, S., 2014. Biosorption of lead (II) ions on Sargassum ilicifolium:Application of response surface methodology. Int. Biodeterior. Biodegrad. 93, 145e152. Van der Ent, A., Baker, A.J.M., Reeves, R.D., Pollard, A.J., Schat, H., 2012. Hyperaccumulators of metal and metalloid trace elements: facts and fiction. Plant Soil 362, 319e334. Wu, C., Zou, Q., Xue, S.G., Pan, W.S., Huang, L., Hartley, W., Mo, J.Y., Wong, M.H., 2016. The effect of silicon on iron plaque formation and arsenic accumulation in rice genotypes with different radial oxygen loss (ROL). Environ. Pollut. 212, 27e33. Xu, X., Huang, Q., Huang, Q., Chen, W., 2012. Soil microbial augmentation by an EGFP-tagged Pseudomonas putida X4 to reduce phytoavailable cadmium. Int. Biodeterior. Biodegrad. 71, 55e60. Xue, S.G., Zhu, F., Kong, X.F., Wu, C., Huang, L., Huang, N., Hartley, W., 2016a. A review of the characterization and revegetation of bauxite residues (Red mud). Environ. Sci. Pollut. Res. 23, 1120e1132. Xue, S.G., Zhu, F., Wu, C., Lei, J., Hartley, W., Pan, W.S., 2016b. Effects of manganese on the microstructures of Chenopodium ambrosioides L., a manganese tolerant plant. Int. J. Phytoremediation 18, 710e719. Yin, Y., Hu, Y., Xiong, F., 2011. Sorption of Cu(II) and Cd(II) by extracellular polymeric substances (EPS) from Aspergillus fumigatus. Int. Biodeterior. Biodegrad. 65, 1012e1018. Yu, X., Gu, J.D., 2007. Accumulation and distribution of trivalent chromium and effects on hybrid willow (Salix matsudana koidz alba L.) metabolism. Archives Environ. Contam. Toxicol. 52, 503e511. Yu, X., Gu, J.D., 2008. Differences in uptake and translocation of hexavalent and trivalent chromium by two species of willows. Ecotoxicology 17, 747e755. Zhu, F., Xue, S.G., Hartley, W., Huang, L., Wu, C., Li, X.F., 2016. Novel predictors of soil genesis following natural weathering processes of bauxite residues. Environ. Sci. Pollut. Res. 23, 2856e2863. Zhou, Q., Diao, C., Sun, Y., Zhou, J., 2012. Tolerance,uptake and removal of nitrobenzene by a newly-found remediation species Mirabilis jalapa L. Chemosphere 86, 994e1000.
Please cite this article in press as: Wang, J., et al., The physiological response of Mirabilis jalapa Linn. to lead stress and accumulation, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.04.030
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
The physiological response of Mirabilis jalapa Linn. to lead stress and accumulation Jun Wang a, Sheng Ye b, Shengguo Xue a, *, William Hartley c, Hao Wu a, Lizheng Shi a a
School of Metallurgy and Environment, Central South University, Changsha 410083, PR China Jiangsu Sentay Environmental Science and Technology Co., Ltd., Nanjing 211153, PR China c Crop and Environment Sciences Department, Harper Adams University, Newport, Shropshire, TF10 8NB, United Kingdom b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2016 Received in revised form 13 April 2016 Accepted 23 April 2016 Available online xxx
Smelters, metal refineries and mining operations have all been considered as major sources of metal release into the environment. As a highly toxic and cumulative poison, once in the environment Pb is difficult to remove and can adversely affect human health. Mirabilis jalapa Linn. (The Marvel of Peru) is a fast growing plant that shows potential for phytostabilization of Pb contaminated soils. Fourier Transform Infrared (FTIR) spectrometry was adopted to detect physiological changes in the chemical composition of M. jalapa exposed to six different concentrations of Pb in solution (0, 50, 100, 200, 500 and 1000 mmol L1 Pb). Results indicated that biomass was reduced in plants grown in Pb treatments compared to controls, although M. jalapa grew typically well at the greatest concentration, 1000 mmol L1. The concentration of Pb in plant tissues occurred in the order roots > leaves > stems, with a translocation factor of less than 0.04. The absorbance of dominating bands near 3420, 2920, 1610 and 1060 cm1 firstly increased but then declined in root tissues; the bands respectively corresponding to organic acids, carbohydrate, protein and amino acids. However, no obvious changes were observed in leaves and stems. The results suggest that M. jalapa can reduce transportation of Pb from roots to shoots, subsequently preventing Pb toxicity in shoots. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Mirabilis jalapa Linn. Pb toxicity FTIR Chemical composition Phytostabilization
1. Introduction Lead (Pb) is one of the main heavy metal pollutants in the environment. With the development of modern industry, Pb presents a marked increase in the environment due to mining growth, over-application of chemical fertilizers and vehicle exhaust emissions (Pietrzykowski et al., 2014). As a non-essential element for plants, Pb induces metabolic disorders, inhibits growth and development, and following excessive exposure, even death. Furthermore, Pb threatens human health through food chain transfer. Therefore, treatment of soil contaminated by Pb has become a priority (Li et al., 2013). Smelters, metal refineries and mining operations have all been considered as major sources of heavy metal release into the environment (Yu and Gu, 2008). Mine tailings in particular are difficult to manage due to heavy metal contamination, which makes plant establishment difficult as a result of poor physical soil structure and
* Corresponding author. E-mail addresses: [email protected], [email protected] (S. Xue).
elevated metal toxicity (Xue et al., 2016a; Zhu et al., 2016). Establishment of plant tolerance can reduce heavy metal bioavailability (Van der Ent A et al., 2012; Xue et al., 2016b). Plants can be classified into three main groups according to metal uptake characteristics: (i) Excluders, restrict translocation. (ii) Index plants, uptake and translocation reflect soil metal concentrations. (iii) Accumulators, plants actively concentrate metals in their tissues. There is evidence to suggest that higher plants have mechanisms to protect themselves from metal toxicity. These mechanisms include metal sequestration using organic compounds (metal-binding polypeptides), subcellular compartmentalization (generally removal of metals to the cell vacuole), active metal efflux (excreting metals by active pumps) and organic ligand exudation (organic molecules exuded by root cells) (Komal et al., 2015). Two mechanisms for heavy metal tolerance in plants have been proposed. The first is exclusion, meaning that heavy metals are absorbed by plants but then subsequently detached through active transport or aging of organs in vitro to make them discharge (Kiran and Thanasekaran, 2011). The second mechanism is cumulative, whereby the heavy metal becomes non-bioactive following detoxification. Examples include Pb precipitation on cell walls of
http://dx.doi.org/10.1016/j.ibiod.2016.04.030 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang, J., et al., The physiological response of Mirabilis jalapa Linn. to lead stress and accumulation, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.04.030
2
J. Wang et al. / International Biodeterioration & Biodegradation xxx (2016) 1e4
rice which prevent excessive Pb from being transported into the protoplast (Wu et al., 2016), and removal of Cr from hydroponic solution in hybrid willows without showing detectable phytotoxicity (Yu and Gu, 2007). Lead can also be transferred to the vacuole, so as to reduce its concentration in protoplasts. Organic acids and proteins can precipitate Pb by chelation thereby reducing its free state. Mirabilis jalapa (The Marvel of Peru) demonstrates tolerance, rapid growth, and characteristics of native plants found in China, which is an ideal germplasm resource to restore soils contaminated by heavy metals (Zhou et al., 2012). When plants are stressed by heavy metal contamination a change in growth, development and characteristics of accumulation occurs (Liang et al., 2011; Xu et al., 2012). In this paper, the enrichment concentration and tolerance of lead were investigated on hydroponically grown M. jalapa. Fourier transform infrared spectroscopy was used to determine chemical changes in different tissues and organs of M. jalapa when it was stressed by lead uptake (Bosch et al., 2006; Gu et al., 2009; Ohno et al., 2015). The objectives of the present study were to interpret M. jalapa's physiological response to Pb toxicity and determine if it could tolerate high concentrations of Pb. 2. Materials and methods 2.1. Experiment method M. jalapa seeds, collected from the tailings wasteland at the Xiangtan manganese mine, were sprinkled on sand-filled pots. Following germination (14 days), M. jalapa seedlings of the same size were selected, the roots were gently washed with tap-water and then thoroughly rinsed with deionized water. Plants were then grown in black plastic containers supplied with 20 L of Hoagland nutrient solution and exposed to 6 concentrations of Pb: 0 (control), 50, 100, 200, 500 and 1000 mmol L1, added as Pb(NO3)2(AR). Treatments in a glass house were replicated three times. All solutions were continuously aerated and pH maintained at 4.5 using 0.1 M NaOH or 0.1 M HCl, to ensure that Pb remained stable as Pb2þ in an ionic state. Plants were harvested after 35 days growth. Plant roots were then gently washed with tap water and then subsequently washed with deionized water and finally blotted dry with tissue paper. Plant samples were divided into roots, stems and leaves, which were firstly oven dried at 105 C for 30 min and then at 75 C until a constant weight was achieved (48 h). Fresh weight (hereafter referred to as FW) and dry weight (hereafter referred to as DW) of roots, stems and leaves were recorded. Samples of dry plant material for Pb analysis were ground to a fine powder using a stainless steel grinder, so as to pass through a 200 screen mesh.
using an agate pestle and mortar. Each sample was thoroughly mixed with sodium bromide particles and tableted into three almost transparent wafers. The samples were scanned 32 times and the three replicate spectra averaged to account for within sample variability and differences in particle size and packing density. 2.4. Statistical analysis All analyses were performed in quintuplicate. The data were statistically treated with Microsoft Excel 2003, SPSS version 19.0 and Origin 8.0. In the case of homogeneity, Duncans post hoc test was used. If there was no homogeneity, Dunnetts T3 test was performed. All figures were constructed using Origin 8.0. 3. Results and discussion 3.1. Growth response of M. jalapa to Pb concentrations M. jalapa grew normally under the range of Pb concentrations. However, at high concentrations (1000 mmol L1), the leaves rolled up slightly and the roots became black. Nevertheless, the different concentrations of Pb did not affect biomass production (Fig. 1). Therefore, a number of concentrations of Pb played a small role in M. jalapa growth and M. jalapa has a strong Pb tolerance ability in the growth medium. 3.2. Pb uptake and accumulation characteristics of M. jalapa With an increase in Pb concentration, Pb accumulation increased in all parts of the plant (Table 1). The content of Pb was found in the order roots > leaves > stems. Even with different Pb concentrations, the content of Pb in stems was the same as that in the control (P > 0.05). However, Pb increased significantly when plants were exposed to high concentrations of 500 and 1000 mmol L1 (P < 0.05) (see Table 2). Translocation factor (Hereafter referred to as TF) reflects the transportation and distribution of metals in plants from below ground to above. A number of studies (Du et al., 2013; Tabaraki et al., 2014; Yin et al., 2011) have shown that Pb mostly accumulates in the roots, which therefore reduces transportation to the above ground tissues, which is one of the important mechanisms to reduce toxicity and has been demonstrated in this study. No clear trend was observed in the TF of M. jalapa stem and leaf tissues.
2.2. Pb content analysis Subsamples of dried plant tissue (c. 0.15 g) were digested with 90 mL of concentrated HNO3 (AR, mass fraction ¼ 65%), 30 mL of HCl (AR, mass fraction ¼ 36e38%) and 3 mL of HClO4 (AR, mass fraction ¼ 70e72%) in a block heater. Lead was determined using ICP-OES (Jones et al., 2011). 2.3. FTIR analysis The spectral information of various tissues and organs was characterized using Fourier transform infrared (FTIR) spectroscopy in the mid-IR range with a Nicolet IS10 infrared spectrometer. The detector was deuterated triglycine sulphate (DTGS). The measured wavebands ranged from 4000 to 500 cm1 with a resolution of 1 cm1. Plant samples were finely ground with KBr (0.5/50 mg)
Fig. 1. Effects of various concentrations of Pb on the biomass of M. jalapa.
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3.3. FTIR analysis of M. jalapa
Table 1 Effect of various concentrations of Pb on its accumulation in M. jalapa. Concentration/mmol$L1 Pb accumulation/mg kg1
0(CK) 50 100 200 500 1000 Significance level
3
Root
Stem
Leaf
22.9 ± 17.0a 3292.1 ± 959.3b 3968.9 ± 536.3bc 5228.5 ± 1294.1bc 6725.2 ± 128.7bc 9313.3 ± 4344.2c <0.05
3.6 ± 1.1a 45.2 ± 5.1a 74.3 ± 27.7ab 94.2 ± 26.7abc 123.6 ± 103.8bc 247.7 ± 198.3c <0.05
4.4 ± 1.5a 58.6 ± 2.9a 138.4 ± 49.4b 143.6 ± 29.5b 212.6 ± 13.2c 263.1 ± 68.5c <0.05
Table 2 Translocation factor of Pb in M. jalapa at various concentrations of Pb. Concentration/mmol L1
TF in stem
TF in leaf
50 100 200 500 1000
0.014 0.019 0.018 0.018 0.027
0.018 0.035 0.027 0.032 0.028
TF: Translocation factor.
However, the TF was less than 0.04, as Pb is difficult to be transport to above ground tissues. The possible reason is Pb adsorption, sedimentation and passivation, which then accumulated in the roots of M. jalapa. In addition, this demonstrates that M. jalapa has a very strong Pb tolerance. At the same time, TF of Pb accumulation in the leaves is higher than that in the stems with various concentrations of Pb.
In Fig. 2A, the stretching vibration peak of 3420 cm1 (free hydroxyl) is mainly reflected in root carbohydrate (cellulose, hemicelluloses, polysaccharides) (Ren et al., 2008). The peak of absorbance increases with the highest peak at 200 mmol L1, and then the absorbance of the peak reduces with an increase in the concentration of Pb. This suggests that low concentrations of Pb promote the secretion of carbohydrates in M. jalapa roots, while high concentrations of Pb inhibit the secretion of carbohydrates. As Pb concentrations continued to increase, root epidermal cell walls absorb and combine with Pb2þ which then subsequently weakens the hydrogen bonds of the cell wall surfaces. The carboxylic acid OeH and methyl stretching vibration peaks overlap near 2920 cm1, which is mainly from various vitamins, membrane and cell wall components. Absorbance increased in the medium and low Pb concentrations, and then decreased with an increase in Pb concentrations (Fig. 2A). It may be that in the medium and low concentrations, M. jalapa roots continuously secrete chelating organic acids as a mechanism of resistance increasing the carboxylic acid bands. Increasing Pb concentrations may lead to physiological and biochemical imbalances, which may have had an adverse effect on the plants resistance mechanisms and resulted in reduced secretion of organic acids. The bending vibration peak of NeH amide near 1655 cm1e1590 cm1 is characteristic of protein (Ren et al., 2008). Amino acid, peptide and protein substances are also root exudates produced in response to Pb toxicity (Li et al., 2013). In medium and low Pb concentrations, Pb stimulated the synthesis and secretion of these substances to complex Pb and hence reduce Pb toxicity to the plants. However, increased concentrations of Pb may have inhibited the synthesis of these substances. Fig. 2A reveals that the peak near 1060 cm1 is a stretching vibration peak of alcohol and ether-based ester or phenol group CeO
Fig. 2. Absorption FTIR spectra in M. jalapa. A: in roots of M. jalapa; B: in stems of M. jalapa; C: in leaves of M. jalapa. a: 0(CK); b: 50 mmol L1; c: 100 mmol L1; d: 200 mmol L1; e: 500 mmol L1; f:1000 mmol L1.
Please cite this article in press as: Wang, J., et al., The physiological response of Mirabilis jalapa Linn. to lead stress and accumulation, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.04.030
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J. Wang et al. / International Biodeterioration & Biodegradation xxx (2016) 1e4
bond. With lower Pb concentrations, a possible reason for the increase in this peak may be synthesis of these compounds by the plant production of hydroxyls that chemically absorbed Pb2þ. However, when Pb continued to increase, transportation of these compounds to the roots was affected and peak height therefore reduced. Fig. 2B displays the IR spectra of M. jalapa stems. The peak near 1060 cm1 is a stretching vibration peak of alcohol and ether-based, ester or phenol groups. The peak near 1630 cm1 is amino acid, peptide and protein substances of bending vibration amide NH peaks. The peak near 2920 cm1 is a carboxylic acid-type OeH stretching vibration peak of organic acids substances. The peak near 3420 cm1 are carbohydrates (cellulose, hemicelluloses, polysaccharides substances). Fig. 2B reveals that no obvious changes were observed in the remaining peaks except that irregular changes occurred in the 3420 cm1 peak. As the Pb concentration increased, organic components did not change significantly and therefore at a certain range of external Pb concentration, physiological and biochemical processes were less affected, which indicates that M. jalapa possesses strong Pb tolerance. Lead concentration in M. jalapa stems did not change significantly with the increase in Pb concentration. Additionally, this shows that Pb in M. jalapa stem had been impeded. Fig. 2C displays IR spectra from M. jalapa leaves grown in different Pb concentrations. The peak near 3420 cm1 is an intermolecular hydrogen bond OeH stretching vibration of free hydroxyls. At 2920 cm1 a carboxylic acid OeH stretching vibration peak overlapping with a methyl stretching vibration is revealed. The peak near 1650 cm1 is an amide NeH bending vibration peak and near 1380 cm1 are oils and fats containing compounds of methyl CeH deformation vibration peaks. The peak near 1060 cm1 is alcohol- and ether-based, ester or a phenol group of the CO stretching vibration peak. Based on Fig. 2C, the peak heights indicate tolerance characteristics of M. jalapa to Pb. The physiological and biochemical processes of M. jalapa leaves appear not to have been affected have not been significantly affected. There are some reports indicating that heavy metals can induce protein synthesis, such as proline-rich protein, disease-related protein and histidine-rich protein (Gu et al., 2009). Besides it is believed that these heavy metalinduced proteins may have the capability to protect plant cells from heavy metal poisoning (Bosch et al., 2006). The bending vibration peak height of NeH amide near 1650 cm1 was higher when the Pb concentrations increased, which may be related to increased amino acid, peptides and protein production in M. jalapa leaves. 4. Conclusions M. jalapa is resilient to Pb, and was not affected when the concentration of Pb reached 1000 mmol L1. Lead accumulated in the roots, thereby reducing its toxicity to the aerial parts of the plant. FTIR studies demonstrated that there were fluctuations in the production of carboxylic acids, carbohydrates, proteins and amino acids in its roots. These increased with low concentrations of Pb but decreased with higher concentrations. The absorbance of dominating bands near 3420, 2920, 1610 and 1060 cm1 firstly increased but then declined in root tissues; these bands respectively corresponding to organic acids, carbohydrate, protein and amino acids. However, no obvious changes were observed in leaves and stems. Due to M. jalapa's large biomass and resistance to Pb toxicity, it
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Please cite this article in press as: Wang, J., et al., The physiological response of Mirabilis jalapa Linn. to lead stress and accumulation, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.04.030