Bull Environ Contam Toxicol (2014) 92:745–751 DOI 10.1007/s00128-014-1252-3
Accumulation and Localization of Cadmium in Potato (Solanum tuberosum) Under Different Soil Cd Levels Zhifan Chen • Ye Zhao • Lei Gu • Shuifeng Wang Yongliang Li • Fangli Dong
•
Received: 18 June 2013 / Accepted: 7 March 2014 / Published online: 29 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Phytoavailability and uptake mechanism of Cd in edible plant tissues grown on metal polluted agricultural soils has become a growing concern worldwide. Uptake, transport, accumulation and localization of cadmium in potato organs under different soil Cd levels were investigated using inductively-coupled plasma mass spectrometry and energy dispersive X-ray microanalysis. Results indicated that Cd contents in potato organs increased with increasing soil Cd concentrations, and the order of Cd contents in different organs was leaves [ stems/roots [ tubers. Root-to-stem Cd translocation coefficients ranged from 0.89 to 1.81. Cd localization in potato tissues suggested that leaves and stems should be the main compartment of Cd storage and uptake. Although low concentrations of Cd migrated from the root to tuber, Cd accumulation in the tuber exceeded the standard for food security. Therefore, the planting of potato plants in farmland containing Cd should be closely evaluated due to its potential to present health risks. Keywords Phytoavailability Energy dispersive X-ray microanalysis Tuber crops
Z. Chen (&) L. Gu F. Dong Institute of Resources and Environment, College of Environment and Planning, Henan University, Kaifeng 475004, China e-mail: [email protected] Y. Zhao School of Environment, Beijing Normal University, Beijing 100875, China S. Wang Y. Li The Center of Analysis and Testing, Beijing Normal University, Beijing 100875, China
Cadmium pollution in agricultural farmland has become a serious problem in many parts of the world, due to industrial development and the overuse of chemical fertilizers and pesticides which produce serious threats to human health through the food chain (Liu et al. 2003). There is a concern that consumption of foods containing relatively high levels of Cd2? may lead to chronic toxicity (Goncalves et al. 2009; Gallego et al. 2012). Vegetable foods are the most important source of nonoccupational exposure to Cd2? for humans (Satarug et al. 2003), so it is necessary to study Cd2? accumulation and its mechanisms in crops such as potato. Potato (Solanum tuberosum L.) is rich in starch and protein, making it the fourth major crop contributing to the world’s food requirement, after corn, wheat and rice (Chatterjee et al. 2006). It is also the most economically important non-grain field crop (Conn and Cochran 2006). Potato plants are often exposed to various types of environmental agents, either accidentally, by compounds present in polluted air, soil or water, or deliberately, as in the case of agricultural pesticides and plant growth regulators (Goncalves et al. 2009). Knowledge of Cd distribution in plant organs and cell compartments is important to better understand accumulation and tolerance mechanisms in plant species. Studies on Cd localization in plants, especially at the cellular level, have recently attracted the attention of many scientists. New and more technically advanced methods of metal detection and sample preparation are still being developed. Among them, energy dispersive X-ray microanalysis (EMAX) following glutaraldehyde fixation with sulphide supplement or freezing of plant samples, is frequently used. It allows precise localization of many elements in the cell compartments, although is not always useful at very low metal concentrations (Wojcik and Tukiendorf 2005). There are different methods for determining element distribution
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in plant tissues such as histochemical (Seregin and Ivanov 1997), cell fractionation (Lozano Rodriguez et al. 1997), X-ray microanalysis (Khan et al. 1984), particle-induced X-ray emission (micro-PIXE) (Ager et al. 2003) or nuclear micro-probe technique (NMP) (Ager et al. 2003). Histochemical detection, using the silver sulfide staining method, is highly sensitive and allows detection of very low metal concentrations both in light and electron microscopy. However, the reaction is not specific, and positive results are obtained with not only Cd sulfide but also with Fe, Zn or Cu. This makes it impossible to distinguish between the deposits of these metals. On the other hand, X-ray microanalysis, although not always useful at very low metal concentrations, provides more specific signals which is why the method is being used more often (Wojcik et al. 2005). EMAX is a widely used tool employed to detect elemental composition and its spatial distribution in a sample without causing damage (Wang 2006). Some studies concerning the effect and accumulation of Cd2? on the potato have been conducted. For instance, Baghour et al. (2001) studied phytoextraction of Cd and Pb, as well as physiological effects in potato plants based on root temperature condition. Maier et al. (2002) studied effect of nitrogen source and calcitic lime on soil pH and potato yield, leaf chemical composition, and tuber Cd concentrations. Dunbar et al. (2003) studied the uptake and partitioning of Cd in two cultivars of potato. In Reid et al.(2003), the mobility of Cd in potato plants was examined using both short-term radioisotopic labeling with Cd-109 and long-term growth experiments in soil supplemented with Cd. Few data on accumulation mechanisms and localization interpretation in potato organs exposed to the Cd stress is available. Therefore, the objective of the current research is to investigate Cd uptake, translocation, accumulation and localization in the tissue and cellular levels of potato organs exposed to different Cd soil concentrations through combining inductively-coupled plasma mass spectrometry (ICP-MS) with EMAX. Additionally, a discussion on the possible tolerance and bioaccumulation mechanisms of potato on Cd will be made. Possible food security risks of potato planted on low-level Cd contaminated soil will also be considered.
Bull Environ Contam Toxicol (2014) 92:745–751
through a 2 mm sieve. Samples of 2 kg of soil were amended with CdCl2 to achieve the concentrations of 0, 1, 5, and 25 mg kg-1 of Cd. All treated soils were transferred into plastic pots to conduct the greenhouse experiment. Eight uniform and well tubers were sown directly into the soil. All treatments were set up in triplicate. Pots were watered to 60 % of the soil water holding capacity with deionized water on a daily basis by weighing the pots and adding water to compensate for any weight loss. When harvested, plants were uprooted from the soils carefully, and separated into roots, stems, leaves and tubers. After rinsing with deionized water in an ultrasonic cleaner, plant samples were dried in an oven at 65°C for 12 h and then reduced to a fine powder in a porcelain mortar for Cd measurement. Precisely weighed quantities of each sample (0.2000 g) were used to prepare the solution in a microwave digester. A mixture of nitric acid (2 mL) at 65 % and hydrogen peroxide (1 mL) at 30 % was used. Extracted solutions, clear and free of organic substances, were left to cool and their volume was adjusted to 10 mL with double-distilled water. Quantitative analysis of Cd in the solutions was carried out with ICP-MS (Element 2, Finnigan Co., Waltham, MA, USA). The detection limit of Cd was 3.13 9 10-12. Alongside the preparation of the sample solutions, for every digestion cycle, a blank was prepared. All sample determinations were repeated three times to minimize the risk of error. Fresh tubers and stem sections (about 1–3 mm in length) and leaf sections (*1 mm2) from the middle section of
Materials and Methods Seedlings were developed from tubers of potato in polyethylene pots with 2 kg soil. Soil used in this study, classified as Calcaric Cambisol, was collected at the 0–30 cm depth. Average selected chemical and physical properties of the soil were pH 8.2, CaCO3 2.73 %, TP 87.6 %, organic carbon 14.6 g kg-1. Soil was air dried and passed
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Fig. 1 Cd contents and BCFs in potato organs with different soil Cd treatments (0, 1, 5, 25 mg kg-1). Cd concentration value in potato organs is the mean ± SD of three individual replicates. Each BCF value of Cd equals to the ratio of the average Cd concentration in the plants to Cd concentration in the soil
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T1
T0
Ep Co
T1-1(0.00, 0.00)
Co T1-2(1.21, 0.20)
T2
T3 T3-1(0.02, 0.00)
CB
T2-3(0.00, 0.00)
T3-2(0.29, 0.03) T2-1(0.21, 0.02) T2-2(0.08, 0.01)
T3-3(0.04, 0.01)
T4
Pm
T5
Pm
T5-2(0.06, 0.01)
T4-2(0.08, 0.01)
T4-1(0.00, 0.00) T5-1(0.09, 0.01)
SG
CW
Fig. 2 The localization of Cd in the cells of the tuber of the potato exposed to 25 mg kg-1 soil Cd. Arrows indicated localization of Cdrich sites. Ep epidermis, Co cortex, SG starch granule, CW cell wall,
Pm plasmalemma, CB cytoplasmic band. Data shown in parentheses are wt% and at.% of Cd, respectively
well-grown leaves of potato plant treated with the highest metal concentration were selected for EMAX studies. Based on previous studies (Wojcik et al. 2005; Wojcik and Tukiendorf 2005; Islam et al. 2008; Jin et al. 2008), an improved method of sample preparation for electron microscopy analysis and Cd localization by EMAX was applied. Root, stem and leaf sections were fixed in 2.5 % glutaraldehyde (v/v) in 0.135 M sodium phosphate buffer (pH 7.35) for 6–8 h at 4°C. To precipitate metals in tissues
and prevent their washing out or dislocation during further preparation procedure, the fixing mixture Na2S (1 %, w/v) was added (Sarret et al. 2001). Samples were then rinsed in fresh buffer and dehydrated in an ascending series of ethanol (30 %, 50 %, 70 %, 80 %, 90 %, 95 %, and 100 %). After dehydration, the preparations were freeze-dried. Dried plant material was cut into thin sections (100 nm) with diamond knives. Specimens were used for X-ray microanalysis in a cold field emission scanning electron
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S0
S1
E Co
Ph Ca
S1-1(0.00, 0.00)
Co
Xy S1-4(0.06, 0.01)
S1-2(0.00, 0.00)
S1-3(0.16, 0.02)
S2
Xy
S3
S2-2(0.06, 0.01)
Ca Co Ph
S3-2(0.00,0.00)
S2-1(0.15,0.02)
E
S3-1(0.06,0.01)
Fig. 3 The localization of Cd in the stem of a potato exposed to 25 mg kg-1 soil Cd. E epidermis, Co cortex, Ph phloem, Xy xylem, Ca cambium
microscope (SEM, S-4800, Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with an energy dispersive X-ray microanalyser (EDXA 350). Microanalysis was performed at an accelerating voltage of 20 kV with a take-off angle of 38°. Spectra from 0 to 20 keV with Cd (L) peak was recorded after 60 s. Detection limit was 0.1 %–0.5 %.
Results and Discussion As presented in Fig. 1, Cd accumulation in potato organs (tuber, root, stem and leaf) increased with increasing Cd contents in soils. Cd concentrations in different organs varied greatly in the order of leaves [ stems/roots [ tubers. Under different soil Cd treatments (0, 1, 5and 25 mg kg-1), the average Cd contents in potato leaves, were 1.31, 11.53, 51.84 and 75.73 mg kg-1, respectively, and for roots (stems), its contents were 0.5 (0.9), 5.03 (7.69), 24.62 (24.04) and 40.71 (41.37) mg kg-1, respectively. Bioconcentration factors (BCFs) of Cd in roots, stems and leaves of potato decreased with increasing Cd content in soils and ranged from 5.03 to 1.63 for roots, 7.69 to 1.65 for stem, and 11.53 to 3.03 for
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leaves. Root-to-stem Cd translocation coefficients (TCs) ranged from 0.89 to 1.81, and TC from root-to-leaf ranged from 1.81 to 3.28. Potato presented good phytoextraction ability for soil Cd. Note that there was very low Cd concentration in tubers, the average Cd contents under various soil Cd concentrations applied (0, 1, 5 and 25 mg kg-1) were 0.11, 0.81, 3.62 and 4.92 mg kg-1, respectively. Results were in agreement with Dunbar et al. (2003) and Reid et al. (2003). These studies showed that tubers contribute only a minor fraction to the overall Cd absorption by the potato, and Cd absorbed from the soil was mainly translocated to the upper parts of the plants, with high concentrations in stems and leaves. This might be attributed to the transpiration of leaves and short growth period of tubers. Growth of tubers didn’t always begin until the growth telophase of buds (Li 1991). However, even in control soils (about 0.258 mg kg-1), Cd concentrations in tubers was greater than the European Commission (EC) (2006) limit and Ministry of Health P. R. China (MOH) limit (0.1 mg kg-1) (SEPA 2005), which was implied that when soil Cd concentration [0.258 mg kg-1, potential risks exist for consumption of potatoes grown under such conditions.
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Results demonstrated a high Cd accumulation in the leaves of the potato and very low contents in the tubers (Fig. 1). Therefore, cellular distribution of Cd in different organs was analyzed in potato plants exposed to a Cd concentration of 25 mg kg-1. Figure 2 presents the SEM cross-section of a potato tuber, as well as wt% and at.% of Cd in localization sites. Granular white deposits were observed in many parts inside the cortex parenchyma cells (T1-2), the surface of global or elliptic starch granules (T21, T2-2, T3-1 and T3-3), or the interior surface of the plasmalemma in the protoplast (T4-2, T5-1 and T5-2) and cytoplasmic band (T3-2), whereas in the epidermis (T1-1) and central cell walls (T4-1) in the tubers, deposits remained undetectable. Results suggested that Cd2? could enter into cells and become a part of cytoplasm through plasmalemma. However, cells in the epidermis, cortex and the central cell walls in the tubers might be a barrier to Cd2?. Yao et al. (2012) indicated that excess Mn in the chloroplast was detoxified through its deposition in a starch granule, which served as a novel detoxifying strategy. Thus, starch granules in the potato might be coordinated with Cd2? to reduce the toxicity of Cd to the tuber. Moreover, Fu et al. (2011) deduced that the vacuole was
L0
the predominant sink for Cd. Localization of Cd in the tuber’s cells (Fig. 2) indicates that the Cd is likely coordinated with the sulfur-rich peptides and organic acids of the cell sap in the vacuole of the tuber (Monteiro et al. 2009). However, TC values corresponding to the transport from root to tuber were the lowest TCs, suggesting a significant amount of Cd likely does not move from root to tuber because sulfhydryl binding or/and complexation to other constituents of the sieve tubes can easily reduce the mobility of Cd from the phloem to the tuber (Reid et al. 2003). The SEM cross-section of potato stem, as well as wt% and at.% of Cd in localization sites were presented in Fig. 3. Large amounts of Cd were detected in the cortex (S1-3, S1-4 and S2-1) and the adjacent phloem (S2-2 and S3-1), which suggests that a proportion of Cd might be transported from the xylem to the phloem. This result was in agreement with the finding reported by Reid et al. (2003), which indicated that routeway and storage functions of pith ray play an important role in the transfer from the xylem to the phloem. Thus, it could be speculated that the cambium between xylem and phloem of stems should have an important role in transfer and storage of Cd. In
H
L1-2(0.00, 0.00)
L1
Vb
L1-1(0.00, 0.00)
L1-3(0.00, 0.00)
L2
L3-1(0.38, 0.05)
L2-1(0.00, 0.00) L3-2(0.00, 0.00)
L3
H Ep
L2-2(0.04, 0.01) L3-3(0.27, 0.05)
Pl
Ec L3-4(0.91, 0.12)
GC
Sg S L2-3(0.08, 0.01)
ep
Fig. 4 Distribution of Cd in the leaf’s tissues of a potato exposed to 25 mg kg-1 soil Cd. H epidermal hair, Ep upper epidermis, Pl palisade tissue, Sg spongy tissue, ep lower epidermis, Vb leaf vein, S stoma, GC guard cell, Ec epidermis cell
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addition, Cheng and Bing (2002) illustrated the main function of the phloem was to transport organic matter generated through photosynthesis. Cd was detected in the phloem by EDXA. Analysis of the EDXA spectrum revealed that deposits of Cd contained sulfur and phosphorus. The complexation of Cd might be ascribed to two parts: Cd complexation with the products of photosynthesis and Cd coordination with the compartments of the cell walls (including cellulose, hemicelluloses, and pectin) (Fu et al. 2011). Cd complexation could reduce the migration of Cd from the above-ground portions of the potato to the tuber and then decrease the toxicity of Cd to the tuber of the potato. Figure 4 presents the fragments of potato leaf made from SEM, as well as wt% and at.% of the main elements including Cd in localization sites. Cd was mainly distributed in the leaf tissues of the potato and outside of the leaf’s stoma (Fig. 4). White deposits of Cd were detected in the palisade and spongy mesophyll cells (L3-3 and L34), the stoma (L2-2), and the adjacent outside of the stoma (L2-3 and L3-1). It was found that specific Cd localization in mesophyll cells lie on the way of water migration from vascular cylinder to epidermis and stomata distinctly, which in agreement with the finding reported by Wojcik et al. (2005). This result indicates the transpiration stream in leaves led to passive Cd transport. In conclusion, current results showed that potato could effectively absorb Cd from soils. The BCFs of Cd in different organs ranged from 0.20 to 0.81 for tubers, 1.63 to 5.03 for roots, 1.65 to 7.69 for stem, and 3.03 to 11.53 for leaves. Correspondingly, the order of Cd concentration in each potato organ was leaves [ stems/roots [ tubers. Mechanisms of Cd uptake and accumulation seem to be localized in leaves and stems. The strong transpiration of leaves help to uptake and accumulate large amounts of Cd, which led to its highest Cd concentration. In addition, our research showed that potato might endure low soil Cd concentration (lower than 25 mg kg-1). Both in tubers, stems and leaves, Cd detected inside cells occurred in the form of electron-dense granules. Cd-bearing electron-dense granules were suggested to represent a less toxic storage form of Cd (Rauser and Ackerley 1987). However, the nature of these deposits remains unknown. Cd might be associated in them with phytochelatins, organic acids or phytate. Accumulation of phytochelatins was the common and main mechanism of Cd detoxification in plants (Wojcik et al. 2005). Hence, further research (using other techniques, such as l-EXAFS) was necessary to reveal the true chemical identity of these granular deposits. At the same time, the low TC values were obtained from the root to the tuber, and the concentration of Cd in the tuber exceeded the food security risk for humans. Therefore, the planting of potato in farmland containing a high level of Cd
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should be carefully evaluated to avoid the food security risk to humans associated with Cd toxicity. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 41301336) and the key science and technology research projects of the Education Department of Henan province, China (Grant No. 13A610064).
References Ager FJ, Ynsa MD, Dominguez-Solis JR, Lopez-Martin MC, Gotor C, Romero LC (2003) Nuclear micro-probe analysis of Arabidopsis thaliana leaves. Nucl Instrum Methods B 210:401–406 Baghour M, Moreno DA, Villora G, Hernandez J, Castilla N, Romero L (2001) Phytoextraction of Cd and Pb and physiological effects in potato plants (Solanum tuberosum var. Spunta): importance of root temperature. J Agric Food Chem 49:5356–5363 Chatterjee C, Gopal R, Dube BK (2006) Impact of iron stress on biomass, yield, metabolism and quality of potato (Solanum tuberosum L.). Sci Hortic 108:1–6 Cheng XF, Bing YD (2002) Phytology. Zhejiang University Press, Zhejiang Conn JS, Cochran VL (2006) Response of potato (Solanum tuberosum L.) to elevated atmospheric CO2 in the North American subarctic. Agric Ecosyst Environ 112:49–57 Dunbar KR, McLaughlin MJ, Reid RJ (2003) The uptake and partitioning of cadmium in two cultivars of potato (Solanum tuberosum L.). J Exp Bot 54:349–354 European Commission (2006) Maximum levels of contaminants in foods. Heavy metals, vol 1881/2006. European Commission Fu XP, Dou CM, Chen YX, Chen XC, Shi JY, Yu MG, Xu J (2011) Subcellular distribution and chemical forms of cadmium in Phytolacca americana L. J Hazard Mater 186:103–107 Gallego SM, Pena LB, Barcia RA, Azpilicueta CE, Lannone MF, Rosales EP, Zawoznik MS, Groppa MD, Benavides MP (2012) Unravelling cadmium toxicity and tolerance in plants: insight into regulatory mechanisms. Environ Exp Bot 83:33–46 Goncalves JF, Antes FG, Maldaner J, Pereira LB, Tabaldi LA, Rauber R, Rossato LV, Bisognin DA, Dressler VL, Flores EMD, Nicoloso FT (2009) Cadmium and mineral nutrient accumulation in potato plantlets grown under cadmium stress in two different experimental culture conditions. Plant Physiol Biochem 47:814–821 Islam E, Lisu D, Li TQ, Yang X, Jin XF, Mahmood Q, Tian SK, Li JY (2008) Effect of Pb toxicity on leaf growth, physiology and ultrastructure in the two ecotypes of Elsholtzia argyi. J Hazard Mater 154(1–3):914–926 Jin XF, Yang XO, Islam E, Liu D, Mahmood Q (2008) Effects of cadmium on ultrastructure and antioxidative defense system in hyperaccumulator and non-hyperaccumulator ecotypes of Sedum alfredii hance. J Hazard Mater 156(1–3):387–397 Khan DH, Duchett JG, Frankland B, Kirkham JB (1984) An X-ray microanalytical study of the distribution of cadmium in roots of Zea mays L. J Plant Physiol 115:19–28 Li Y (1991) Dissection and technology of vegetables. Agricultural Press, Beijing Liu J, Li KQ, Xu JK, Liang JS, Lu XL, Yang JC, Zhu QS (2003) Interaction of Cd and five mineral nutrients for uptake and accumulation in different rice cultivars and genotypes. Field Crop Res 83:271–281 Lozano Rodriguez E, Hernandez LE, Bonay P, CarpenaRuiz RO (1997) Distribution of cadmium in shoot and root tissues of maize and pea plants: physiological disturbances. J Exp Bot 48(306):123–128
Bull Environ Contam Toxicol (2014) 92:745–751 Maier NA, McLaughlin MJ, Heap M, Butt M, Smart MK (2002) Effect of nitrogen source and calcitic lime on soil pH and potato yield, leaf chemical composition, and tuber cadmium concentrations. J Plant Nutr 25:523–544 Monteiro MS, Santos C, Soares AMVM, Mann RM (2009) Assessment of biomarkers of cadmium stress in lettuce. Ecotoxicol Environ Saf 72:811–818 Rauser WE, Ackerley CA (1987) Localization of cadmium in granules within differentiating and mature root cells. Can J Bot 65:643–646 Reid RJ, Dunbar KR, McLaughlin MJ (2003) Cadmium loading into potato tubers: the roles of the periderm, xylem and phloem. Plant Cell Environ 26:201–206 Sarret G, Vangronsveld J, Manceau A, Musso M, D’Haen J, Menthonnex JJ, Hazemann JL (2001) Accumulation forms of Zn and Pb in Phaseolus vulgaris in the presence and absence of EDTA. Environ Sci Technol 35(13):2854–2859 Satarug S, Baker JR, Urbenjapol S, Haswell-Elkins M, Reilly PEB, Williams DJ, Moore MR (2003) A global perspective on
751 cadmium pollution and toxicity in non-occupationally exposed population. Toxicol Lett 137:65–83 SEPA (2005) The limits of pollutants in food. GB 2762-2005. Beijing Seregin IV, Ivanov VB (1997) Histochemical investigation of cadmium and lead distribution in plants. Russ J Plant Physiol 44:791–796 Wang X (2006) A chemical signal possibly related to physiology in fossil cells detected by energy dispersive X-ray microanalysis. Tissue Cell 38(1):43–51 Wojcik M, Tukiendorf A (2005) Cadmium uptake, localization and detoxification in Zea mays. Biol Plant 49(2):237–245 Wojcik M, Vangronsveld J, D’Haen J, Tukiendorf A (2005) Cadmium tolerance in Thlaspi caerulescens—II. Localization of cadmium in Thlaspi caerulescens. Environ Exp Bot 53:163–171 Yao Y, Xu G, Mou D, Wang J, Ma J (2012) Subcellular Mn compartation, anatomic and biochemical changes of two grape varieties in response to excess manganese. Chemosphere 89(2):150–157
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Accumulation and Localization of Cadmium in Potato (Solanum tuberosum) Under Different Soil Cd Levels Zhifan Chen • Ye Zhao • Lei Gu • Shuifeng Wang Yongliang Li • Fangli Dong
•
Received: 18 June 2013 / Accepted: 7 March 2014 / Published online: 29 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Phytoavailability and uptake mechanism of Cd in edible plant tissues grown on metal polluted agricultural soils has become a growing concern worldwide. Uptake, transport, accumulation and localization of cadmium in potato organs under different soil Cd levels were investigated using inductively-coupled plasma mass spectrometry and energy dispersive X-ray microanalysis. Results indicated that Cd contents in potato organs increased with increasing soil Cd concentrations, and the order of Cd contents in different organs was leaves [ stems/roots [ tubers. Root-to-stem Cd translocation coefficients ranged from 0.89 to 1.81. Cd localization in potato tissues suggested that leaves and stems should be the main compartment of Cd storage and uptake. Although low concentrations of Cd migrated from the root to tuber, Cd accumulation in the tuber exceeded the standard for food security. Therefore, the planting of potato plants in farmland containing Cd should be closely evaluated due to its potential to present health risks. Keywords Phytoavailability Energy dispersive X-ray microanalysis Tuber crops
Z. Chen (&) L. Gu F. Dong Institute of Resources and Environment, College of Environment and Planning, Henan University, Kaifeng 475004, China e-mail: [email protected] Y. Zhao School of Environment, Beijing Normal University, Beijing 100875, China S. Wang Y. Li The Center of Analysis and Testing, Beijing Normal University, Beijing 100875, China
Cadmium pollution in agricultural farmland has become a serious problem in many parts of the world, due to industrial development and the overuse of chemical fertilizers and pesticides which produce serious threats to human health through the food chain (Liu et al. 2003). There is a concern that consumption of foods containing relatively high levels of Cd2? may lead to chronic toxicity (Goncalves et al. 2009; Gallego et al. 2012). Vegetable foods are the most important source of nonoccupational exposure to Cd2? for humans (Satarug et al. 2003), so it is necessary to study Cd2? accumulation and its mechanisms in crops such as potato. Potato (Solanum tuberosum L.) is rich in starch and protein, making it the fourth major crop contributing to the world’s food requirement, after corn, wheat and rice (Chatterjee et al. 2006). It is also the most economically important non-grain field crop (Conn and Cochran 2006). Potato plants are often exposed to various types of environmental agents, either accidentally, by compounds present in polluted air, soil or water, or deliberately, as in the case of agricultural pesticides and plant growth regulators (Goncalves et al. 2009). Knowledge of Cd distribution in plant organs and cell compartments is important to better understand accumulation and tolerance mechanisms in plant species. Studies on Cd localization in plants, especially at the cellular level, have recently attracted the attention of many scientists. New and more technically advanced methods of metal detection and sample preparation are still being developed. Among them, energy dispersive X-ray microanalysis (EMAX) following glutaraldehyde fixation with sulphide supplement or freezing of plant samples, is frequently used. It allows precise localization of many elements in the cell compartments, although is not always useful at very low metal concentrations (Wojcik and Tukiendorf 2005). There are different methods for determining element distribution
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in plant tissues such as histochemical (Seregin and Ivanov 1997), cell fractionation (Lozano Rodriguez et al. 1997), X-ray microanalysis (Khan et al. 1984), particle-induced X-ray emission (micro-PIXE) (Ager et al. 2003) or nuclear micro-probe technique (NMP) (Ager et al. 2003). Histochemical detection, using the silver sulfide staining method, is highly sensitive and allows detection of very low metal concentrations both in light and electron microscopy. However, the reaction is not specific, and positive results are obtained with not only Cd sulfide but also with Fe, Zn or Cu. This makes it impossible to distinguish between the deposits of these metals. On the other hand, X-ray microanalysis, although not always useful at very low metal concentrations, provides more specific signals which is why the method is being used more often (Wojcik et al. 2005). EMAX is a widely used tool employed to detect elemental composition and its spatial distribution in a sample without causing damage (Wang 2006). Some studies concerning the effect and accumulation of Cd2? on the potato have been conducted. For instance, Baghour et al. (2001) studied phytoextraction of Cd and Pb, as well as physiological effects in potato plants based on root temperature condition. Maier et al. (2002) studied effect of nitrogen source and calcitic lime on soil pH and potato yield, leaf chemical composition, and tuber Cd concentrations. Dunbar et al. (2003) studied the uptake and partitioning of Cd in two cultivars of potato. In Reid et al.(2003), the mobility of Cd in potato plants was examined using both short-term radioisotopic labeling with Cd-109 and long-term growth experiments in soil supplemented with Cd. Few data on accumulation mechanisms and localization interpretation in potato organs exposed to the Cd stress is available. Therefore, the objective of the current research is to investigate Cd uptake, translocation, accumulation and localization in the tissue and cellular levels of potato organs exposed to different Cd soil concentrations through combining inductively-coupled plasma mass spectrometry (ICP-MS) with EMAX. Additionally, a discussion on the possible tolerance and bioaccumulation mechanisms of potato on Cd will be made. Possible food security risks of potato planted on low-level Cd contaminated soil will also be considered.
Bull Environ Contam Toxicol (2014) 92:745–751
through a 2 mm sieve. Samples of 2 kg of soil were amended with CdCl2 to achieve the concentrations of 0, 1, 5, and 25 mg kg-1 of Cd. All treated soils were transferred into plastic pots to conduct the greenhouse experiment. Eight uniform and well tubers were sown directly into the soil. All treatments were set up in triplicate. Pots were watered to 60 % of the soil water holding capacity with deionized water on a daily basis by weighing the pots and adding water to compensate for any weight loss. When harvested, plants were uprooted from the soils carefully, and separated into roots, stems, leaves and tubers. After rinsing with deionized water in an ultrasonic cleaner, plant samples were dried in an oven at 65°C for 12 h and then reduced to a fine powder in a porcelain mortar for Cd measurement. Precisely weighed quantities of each sample (0.2000 g) were used to prepare the solution in a microwave digester. A mixture of nitric acid (2 mL) at 65 % and hydrogen peroxide (1 mL) at 30 % was used. Extracted solutions, clear and free of organic substances, were left to cool and their volume was adjusted to 10 mL with double-distilled water. Quantitative analysis of Cd in the solutions was carried out with ICP-MS (Element 2, Finnigan Co., Waltham, MA, USA). The detection limit of Cd was 3.13 9 10-12. Alongside the preparation of the sample solutions, for every digestion cycle, a blank was prepared. All sample determinations were repeated three times to minimize the risk of error. Fresh tubers and stem sections (about 1–3 mm in length) and leaf sections (*1 mm2) from the middle section of
Materials and Methods Seedlings were developed from tubers of potato in polyethylene pots with 2 kg soil. Soil used in this study, classified as Calcaric Cambisol, was collected at the 0–30 cm depth. Average selected chemical and physical properties of the soil were pH 8.2, CaCO3 2.73 %, TP 87.6 %, organic carbon 14.6 g kg-1. Soil was air dried and passed
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Fig. 1 Cd contents and BCFs in potato organs with different soil Cd treatments (0, 1, 5, 25 mg kg-1). Cd concentration value in potato organs is the mean ± SD of three individual replicates. Each BCF value of Cd equals to the ratio of the average Cd concentration in the plants to Cd concentration in the soil
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T1
T0
Ep Co
T1-1(0.00, 0.00)
Co T1-2(1.21, 0.20)
T2
T3 T3-1(0.02, 0.00)
CB
T2-3(0.00, 0.00)
T3-2(0.29, 0.03) T2-1(0.21, 0.02) T2-2(0.08, 0.01)
T3-3(0.04, 0.01)
T4
Pm
T5
Pm
T5-2(0.06, 0.01)
T4-2(0.08, 0.01)
T4-1(0.00, 0.00) T5-1(0.09, 0.01)
SG
CW
Fig. 2 The localization of Cd in the cells of the tuber of the potato exposed to 25 mg kg-1 soil Cd. Arrows indicated localization of Cdrich sites. Ep epidermis, Co cortex, SG starch granule, CW cell wall,
Pm plasmalemma, CB cytoplasmic band. Data shown in parentheses are wt% and at.% of Cd, respectively
well-grown leaves of potato plant treated with the highest metal concentration were selected for EMAX studies. Based on previous studies (Wojcik et al. 2005; Wojcik and Tukiendorf 2005; Islam et al. 2008; Jin et al. 2008), an improved method of sample preparation for electron microscopy analysis and Cd localization by EMAX was applied. Root, stem and leaf sections were fixed in 2.5 % glutaraldehyde (v/v) in 0.135 M sodium phosphate buffer (pH 7.35) for 6–8 h at 4°C. To precipitate metals in tissues
and prevent their washing out or dislocation during further preparation procedure, the fixing mixture Na2S (1 %, w/v) was added (Sarret et al. 2001). Samples were then rinsed in fresh buffer and dehydrated in an ascending series of ethanol (30 %, 50 %, 70 %, 80 %, 90 %, 95 %, and 100 %). After dehydration, the preparations were freeze-dried. Dried plant material was cut into thin sections (100 nm) with diamond knives. Specimens were used for X-ray microanalysis in a cold field emission scanning electron
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S0
S1
E Co
Ph Ca
S1-1(0.00, 0.00)
Co
Xy S1-4(0.06, 0.01)
S1-2(0.00, 0.00)
S1-3(0.16, 0.02)
S2
Xy
S3
S2-2(0.06, 0.01)
Ca Co Ph
S3-2(0.00,0.00)
S2-1(0.15,0.02)
E
S3-1(0.06,0.01)
Fig. 3 The localization of Cd in the stem of a potato exposed to 25 mg kg-1 soil Cd. E epidermis, Co cortex, Ph phloem, Xy xylem, Ca cambium
microscope (SEM, S-4800, Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with an energy dispersive X-ray microanalyser (EDXA 350). Microanalysis was performed at an accelerating voltage of 20 kV with a take-off angle of 38°. Spectra from 0 to 20 keV with Cd (L) peak was recorded after 60 s. Detection limit was 0.1 %–0.5 %.
Results and Discussion As presented in Fig. 1, Cd accumulation in potato organs (tuber, root, stem and leaf) increased with increasing Cd contents in soils. Cd concentrations in different organs varied greatly in the order of leaves [ stems/roots [ tubers. Under different soil Cd treatments (0, 1, 5and 25 mg kg-1), the average Cd contents in potato leaves, were 1.31, 11.53, 51.84 and 75.73 mg kg-1, respectively, and for roots (stems), its contents were 0.5 (0.9), 5.03 (7.69), 24.62 (24.04) and 40.71 (41.37) mg kg-1, respectively. Bioconcentration factors (BCFs) of Cd in roots, stems and leaves of potato decreased with increasing Cd content in soils and ranged from 5.03 to 1.63 for roots, 7.69 to 1.65 for stem, and 11.53 to 3.03 for
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leaves. Root-to-stem Cd translocation coefficients (TCs) ranged from 0.89 to 1.81, and TC from root-to-leaf ranged from 1.81 to 3.28. Potato presented good phytoextraction ability for soil Cd. Note that there was very low Cd concentration in tubers, the average Cd contents under various soil Cd concentrations applied (0, 1, 5 and 25 mg kg-1) were 0.11, 0.81, 3.62 and 4.92 mg kg-1, respectively. Results were in agreement with Dunbar et al. (2003) and Reid et al. (2003). These studies showed that tubers contribute only a minor fraction to the overall Cd absorption by the potato, and Cd absorbed from the soil was mainly translocated to the upper parts of the plants, with high concentrations in stems and leaves. This might be attributed to the transpiration of leaves and short growth period of tubers. Growth of tubers didn’t always begin until the growth telophase of buds (Li 1991). However, even in control soils (about 0.258 mg kg-1), Cd concentrations in tubers was greater than the European Commission (EC) (2006) limit and Ministry of Health P. R. China (MOH) limit (0.1 mg kg-1) (SEPA 2005), which was implied that when soil Cd concentration [0.258 mg kg-1, potential risks exist for consumption of potatoes grown under such conditions.
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Results demonstrated a high Cd accumulation in the leaves of the potato and very low contents in the tubers (Fig. 1). Therefore, cellular distribution of Cd in different organs was analyzed in potato plants exposed to a Cd concentration of 25 mg kg-1. Figure 2 presents the SEM cross-section of a potato tuber, as well as wt% and at.% of Cd in localization sites. Granular white deposits were observed in many parts inside the cortex parenchyma cells (T1-2), the surface of global or elliptic starch granules (T21, T2-2, T3-1 and T3-3), or the interior surface of the plasmalemma in the protoplast (T4-2, T5-1 and T5-2) and cytoplasmic band (T3-2), whereas in the epidermis (T1-1) and central cell walls (T4-1) in the tubers, deposits remained undetectable. Results suggested that Cd2? could enter into cells and become a part of cytoplasm through plasmalemma. However, cells in the epidermis, cortex and the central cell walls in the tubers might be a barrier to Cd2?. Yao et al. (2012) indicated that excess Mn in the chloroplast was detoxified through its deposition in a starch granule, which served as a novel detoxifying strategy. Thus, starch granules in the potato might be coordinated with Cd2? to reduce the toxicity of Cd to the tuber. Moreover, Fu et al. (2011) deduced that the vacuole was
L0
the predominant sink for Cd. Localization of Cd in the tuber’s cells (Fig. 2) indicates that the Cd is likely coordinated with the sulfur-rich peptides and organic acids of the cell sap in the vacuole of the tuber (Monteiro et al. 2009). However, TC values corresponding to the transport from root to tuber were the lowest TCs, suggesting a significant amount of Cd likely does not move from root to tuber because sulfhydryl binding or/and complexation to other constituents of the sieve tubes can easily reduce the mobility of Cd from the phloem to the tuber (Reid et al. 2003). The SEM cross-section of potato stem, as well as wt% and at.% of Cd in localization sites were presented in Fig. 3. Large amounts of Cd were detected in the cortex (S1-3, S1-4 and S2-1) and the adjacent phloem (S2-2 and S3-1), which suggests that a proportion of Cd might be transported from the xylem to the phloem. This result was in agreement with the finding reported by Reid et al. (2003), which indicated that routeway and storage functions of pith ray play an important role in the transfer from the xylem to the phloem. Thus, it could be speculated that the cambium between xylem and phloem of stems should have an important role in transfer and storage of Cd. In
H
L1-2(0.00, 0.00)
L1
Vb
L1-1(0.00, 0.00)
L1-3(0.00, 0.00)
L2
L3-1(0.38, 0.05)
L2-1(0.00, 0.00) L3-2(0.00, 0.00)
L3
H Ep
L2-2(0.04, 0.01) L3-3(0.27, 0.05)
Pl
Ec L3-4(0.91, 0.12)
GC
Sg S L2-3(0.08, 0.01)
ep
Fig. 4 Distribution of Cd in the leaf’s tissues of a potato exposed to 25 mg kg-1 soil Cd. H epidermal hair, Ep upper epidermis, Pl palisade tissue, Sg spongy tissue, ep lower epidermis, Vb leaf vein, S stoma, GC guard cell, Ec epidermis cell
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addition, Cheng and Bing (2002) illustrated the main function of the phloem was to transport organic matter generated through photosynthesis. Cd was detected in the phloem by EDXA. Analysis of the EDXA spectrum revealed that deposits of Cd contained sulfur and phosphorus. The complexation of Cd might be ascribed to two parts: Cd complexation with the products of photosynthesis and Cd coordination with the compartments of the cell walls (including cellulose, hemicelluloses, and pectin) (Fu et al. 2011). Cd complexation could reduce the migration of Cd from the above-ground portions of the potato to the tuber and then decrease the toxicity of Cd to the tuber of the potato. Figure 4 presents the fragments of potato leaf made from SEM, as well as wt% and at.% of the main elements including Cd in localization sites. Cd was mainly distributed in the leaf tissues of the potato and outside of the leaf’s stoma (Fig. 4). White deposits of Cd were detected in the palisade and spongy mesophyll cells (L3-3 and L34), the stoma (L2-2), and the adjacent outside of the stoma (L2-3 and L3-1). It was found that specific Cd localization in mesophyll cells lie on the way of water migration from vascular cylinder to epidermis and stomata distinctly, which in agreement with the finding reported by Wojcik et al. (2005). This result indicates the transpiration stream in leaves led to passive Cd transport. In conclusion, current results showed that potato could effectively absorb Cd from soils. The BCFs of Cd in different organs ranged from 0.20 to 0.81 for tubers, 1.63 to 5.03 for roots, 1.65 to 7.69 for stem, and 3.03 to 11.53 for leaves. Correspondingly, the order of Cd concentration in each potato organ was leaves [ stems/roots [ tubers. Mechanisms of Cd uptake and accumulation seem to be localized in leaves and stems. The strong transpiration of leaves help to uptake and accumulate large amounts of Cd, which led to its highest Cd concentration. In addition, our research showed that potato might endure low soil Cd concentration (lower than 25 mg kg-1). Both in tubers, stems and leaves, Cd detected inside cells occurred in the form of electron-dense granules. Cd-bearing electron-dense granules were suggested to represent a less toxic storage form of Cd (Rauser and Ackerley 1987). However, the nature of these deposits remains unknown. Cd might be associated in them with phytochelatins, organic acids or phytate. Accumulation of phytochelatins was the common and main mechanism of Cd detoxification in plants (Wojcik et al. 2005). Hence, further research (using other techniques, such as l-EXAFS) was necessary to reveal the true chemical identity of these granular deposits. At the same time, the low TC values were obtained from the root to the tuber, and the concentration of Cd in the tuber exceeded the food security risk for humans. Therefore, the planting of potato in farmland containing a high level of Cd
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should be carefully evaluated to avoid the food security risk to humans associated with Cd toxicity. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 41301336) and the key science and technology research projects of the Education Department of Henan province, China (Grant No. 13A610064).
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