Metal accumulation and detoxification mechanisms in mycorrhizal Betula pubescens

Environmental Pollution xxx (2017) 1e10

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Metal accumulation and detoxification mechanisms in mycorrhizal Betula pubescens*
ndez-Fuego a, b, A. Bertrand a, b, A. Gonza
lez a, b, * D. Ferna a b


tico Rodrigo Uría s/n, 33071 Oviedo, Spain Departamento de Biología de Organismos y Sistemas, Universidad de Oviedo, Catedra Instituto Universitario de Biotecnología de Asturias, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 April 2017 Received in revised form 19 July 2017 Accepted 22 July 2017 Available online xxx

Metal detoxification in plants is a complex process that involves different mechanisms, such as the retention of metals to the cell wall and their chelation and subsequent compartmentalization in plant vacuoles. In order to identify the mechanisms involved in metal accumulation and tolerance in Betula pubescens, as well as the role of mycorrhization in these processes, mycorrhizal and non-mycorrhizal plants were grown in two industrial soils with contrasting concentrations of heavy metals. Mycorrhization increased metal uptake at low metal concentrations in the soil and reduced it at high metal concentrations, which led to an enhanced growth and biomass production of the host when growing in the most polluted soil. Our results suggest that the sequestration on the cell wall is the main detoxification mechanism in white birch exposed to acute chronic metal-stress, while phytochelatins play a role mitigating metal toxicity inside the cells. Given its high Mn and Zn root-to-shoot translocation rate, Betula pubescens is a very promising species for the phytoremediation of soils polluted with these metals. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Heavy metals Mycorrhization Phytochelatins Organic acids Cell wall

1. Introduction Anthropogenic and industrial activities have generated high concentrations of heavy metals in the soil (Kabata-Pendias, 2010; Bonet et al., 2016). The resistance of plants to heavy metals stress relies in part on their ability to tolerate the toxic effects of these elements. At the cellular level, it generally implies maintaining low concentrations of free metal ions in the cytosol through several mechanisms such as the immobilization of the extracellular metal in the cell wall. Many studies have reported the essential role of this structure in metal tolerant and hyperaccumulating plants
ndez (Krzesłowska, 2011; Chen et al., 2013; Sun et al., 2013; Ferna et al., 2014). This metal detoxification mechanism is based on the capacity of different cell wall components (e.g. pectins, phenols or glycoproteins) to bind metals through ionic and non-ionic interactions, which leads to their metabolic inactivation (Zornoza et al., 2002; Krzesłowska, 2011). When the metals reach the cell cytoplasm, the most common

*

This paper has been recommended for acceptance by Prof. W. Wen-Xiong. * Corresponding author. Departamento de Biología de Organismos y Sistemas, Universidad de Oviedo, Catedr
atico Rodrigo Uría s/n, 33071 Oviedo, Spain. E-mail address: [email protected] (A. Gonz
alez).

detoxification strategies adopted by plants is their chelation and subsequent compartmentalization in the vacuole (Corso et al.,
zquez et al., 2009; Krzesłowska, 2011). Low molecular 2005; Va weight ligands such as phytochelatins (PCs) and organic acids (OAs) are important metal chelating compounds. PCs are non-protein thiols (NPTs) enzymatically synthesized from glutathione (GSH), with the basic structure of g-(Glu-Cys)n-Gly (ranging from 2 to 11), which are present in plants, fungi and invertebrates (Zagorchev et al., 2013). Due to their functional thiol groups, NPTs can bind heavy metals, forming less toxic complexes that are later compartmentalized into the cell vacuole (Cobbett and Goldsbrough, 2002). The synthesis of these NPTs is strongly induced as a response to heavy metals in some cases, so they are believed to play an essential role in heavy metal tolerance (Zagorchev et al., 2013). However, there is an increasing debate concerning the importance of this detoxification mechanism especially in metal-tolerant and
ndez metal-hyperaccumulating species (Zhang et al., 2010; Ferna et al., 2014). Besides PCs, small OAs can also bind metals and have also been reported to participate in metal transport, accumulation and detoxification in plants (Sun et al., 2011). A positive correlation between metal exposure and the levels of OAs has been observed in hyperaccumulators (Sun et al., 2011; DalCorso et al., 2013), although their role in metal tolerance and accumulation is controversial since other authors consider that they may not be

http://dx.doi.org/10.1016/j.envpol.2017.07.072 0269-7491/© 2017 Elsevier Ltd. All rights reserved.


ndez-Fuego, D., et al., Metal accumulation and detoxification mechanisms in mycorrhizal Betula Please cite this article in press as: Ferna pubescens, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.07.072


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jcik et al., 2006; Martínezclearly involved in these processes (Wo
ndez et al., 2011). Ferna In natural conditions, the roots of trees associate with soil fungi, forming mycorrhizae, which increase the root absorption area and the uptake of nutrients from the soil. This association is especially important in nutrient-poor or metal-polluted soils since it can improve the survival and development rates of trees growing in these conditions. Some studies have shown that plants associated with metal-tolerant fungal strains perform better at elevated heavy-metal concentrations, generally due to a reduced metal accumulation of the host plant (Krznaric et al., 2009; Colpaert et al., 2011). However, we have observed that mycorrhizal Iberian white birch plants (Betula celtiberica Rothm & Vasc.) had a better development and accumulated more Cd than the non-mycorrhizal plants
ndez et al., 2008). in previous in vitro studies (Ferna Metal detoxification in plants is a complex process with several mechanisms involved. However, the relative importance of each detoxification mechanism is still unclear and depends on many factors, such as the plant species or ecotype, the metal (or metals) involved, its concentration, and, in mycorrhizal plants, the associated fungal species or strain (Jentschke and Goldbold, 2000; Mrnka et al., 2012). Therefore, the aim of this study is to identify the mechanisms involved in heavy-metal tolerance and accumulation in birch plants and their importance, with special focus on the role of the cell wall, PCs and OAs, and the effect of mycorrhization. For this purpose, we selected plants of European white birch (Betula pubescens Ehrh.), a closely related species of B. celtiberica (previously known as B. pubescens subsp. celtiberica) with a wider distribution, and a tolerant strain of the mycorrhizal fungus Paxillus ammoniavirescens Contu & Dessì. Additionally, to evaluate the impact of the soil metal pollution on these processes, plants were grown in two industrial soils with contrasting concentrations of heavy metals: a highly polluted soil (Nitrastur) and a moderately polluted one (Terronal). 2. Materials and methods 2.1. Soil analysis Two different polluted soils were used for this experiment. Terronal soil was collected from a moderately polluted zone of an abandoned mercury mine in Mieres (Asturias, Spain) while Nitrastur soil was collected from an old nitrate factory in Langreo (Asturias, Spain). For measuring the total heavy metal concentration, 7 soil samples per site were collected at a depth of 25e30 cm, mixed before drying in an oven at 35
C for 72 h and filtered through a 2 mm stainless steel sieve. Then, 100 mg of soil sample were digested with 3 mL of highly-purified HNO3 (68%) in a microwave oven and analyzed by inductively coupled plasma-mass
ndez et al. spectrometry (ICP-MS) as previously described in Ferna (2008). To evaluate the fraction of metal in the soil that could be potentially available for plants, the modified three-step sequential extraction procedure proposed by the European Community Bureau of Reference (BCR) was used (Rauret et al., 1999). The certified reference material BCR-701 (Sigma-Aldrich) was used for quality assurance purposes. The resulting fractions were analyzed by ICPMS and the fraction of metal available for plants was calculated as the sum of the exchangeable and reducible fractions, according to Sungur et al. (2014), whereas the non-available fraction corresponded to the sum of the oxidizable and residual fractions. Soil pH was determined by diluting 1.5 g of dry soil in 30 mL of doubled deionized water. Organic matter content was analyzed by combustion and soil texture was determined by laser diffraction spectroscopy using a standardized ISO 13320 (2009) technique that

provides data in the particle size range between 0.017 and 2000 mm. This procedure included disaggregation with the dispersants sodium hexametaphosphate and sodium carbonate (Sierra et al., 2011) and analysis with the Aqueous Liquid Module of the LS 13e320 MW model (Beckman Inc. Coulter). 2.2. Plant material, fungus inoculation and growth conditions Plants of a clone of European white birch (Betula pubescens) were selected based on its high Cd tolerance and accumulation capacity. These plants were originated from seeds of a tree that naturally grew in a metal polluted site. Each germinated seed turned into seedlings that were cloned following the methodology
ndez et al. (2008). Plants were previously described by Ferna cultured in vitro in half-strength Murashige and Skoog medium (pH 5.7) (Murashige and Skoog, 1962), with 0.2 g L1 of sequestrene 138-Fe (Ciba-Geigy AG) as iron source, 30 g L1 of sucrose and 7 g L1 agar. Cultures were kept in a growth chamber at 25
C and 16 h photoperiod for five weeks prior to their inoculation. The ectomycorrhizal fungus Paxillus ammoniavirescens, from a fruitbody collected from a metal-polluted soil in Asturias, was previously selected for its in vitro growth capacity and high Cd accumulation. Plants were inoculated by placing two discs (1 cm diameter) of actively growing fungal mycelium directly on the surface of the plant culture medium. Once plant mycorrhization was observed under a dissection microscope (approximately 4 weeks after inoculation), mycorrhizal (M) and non-mycorrhizal (NM) plants were acclimated to greenhouse conditions. For this purpose, plants were placed in a mist tunnel at 25
C and 90% humidity for two days. Then, plants were transferred to Rootrainer book-like containers (Ronaash, Ltd.) of 20 cm cell depth filled with a sphagnum peat:perlite mixture (3:2, v/v) and kept in mist tunnel for 15 days where the humidity progressively diminished from 90% to 60%. Once the fungal mycelia was well established, plants were transferred to the final culture conditions, 1 L pots (diameter ¼ 14 cm, height ¼ 14 cm) filled with a sphagnum peat:perlite mixture (3:1, v/v) (control) or polluted soils (Terronal and Nitrastur). Both polluted soils had been previously autoclaved twice at an interval of 48 h at 100
C for 1 h, in order to eliminate existing arbuscular mycorrhizal propagules. The experiment was carried out under greenhouse conditions (natural light, 20/30
C temperature and 60e70% of humidity) in a 3  2 completely randomized factorial design (10 plants per treatment). 2.3. Growth parameters After 60 days of culture, plants were carefully extracted from pots and exhaustively rinsed with tap water first, with double deionized water as the final step. Subsequently, their shoot and root length as well as fresh and dry weights were measured. Leaf and root samples were taken in subgroups formed by at least four different plants, homogenized with liquid nitrogen and stored at 80
C until use. 2.4. Extraction of cell walls and metal accumulation The extraction of cell walls was conducted using the protocol
ndez et al. (2014). The resultant supernatant was described in Ferna collected (soluble fraction) and the pellet (cell walls fraction) was oven-dried at 40
C for 48 h. For total metal accumulation, 100 mg of homogenized leaf or root sample were digested with 3 mL of highly-purified HNO3 (68%) and analyzed by ICP-MS as described in section 2.1. To analyze the fractionation of metal in the cell, 100 mg of the supernatant (soluble fraction) or pellet (cell wall fraction) obtained as described above in


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this section, were also analyzed by ICP-MS as described in section 2.1. 2.5. Analysis of non-protein thiols

Table 1 Physicochemical parameters of the soils. Data is expressed as average ± standard error of three different replicates. Parameters

Soil Control

The extraction and analysis of NPTs were carried out from leaf and root samples following the protocol previously described by
ndez et al. (2012). The high-performance liquid chromatogFerna raphy (HPLC) separation was performed using a Waters 600 (Waters Corporation) with a post-column derivatization with Ellman's reagent (Ellman, 1959). The sample (100 mL) was injected into a Kromasil 100 C18 5 mm (250  4.6 mm) (Scharlau) column and eluted with solvent A (acetonitrile: H2O, 2: 98 (v/v) to which 0.05% trifluoroacetic acid (TFA) was added) and solvent B (acetonitrile: H2O, 98: 2 (v/v) also with 0.05% TFA). Samples were separated using a linear gradient (0e25% in 25 min and 25e50% in 5 min) of solvent B at 1.5 mL min1 flow for 30 min. The derivatized thiols were detected at 412 nm using a Waters 996 photodiode array detector and the obtained peaks were identified by comparison with the standards of GSH and a mix of PCs, des-Gly-PCs and Cys-PCs. The quantitative changes in the thiol compounds observed were calculated by the integration of their peak areas at 412 nm of absorbance converted into nmol and quantified as GSH equivalents. 2.6. Analysis of organic acids Organic acids extraction and separation were performed from leaf and root samples following the protocol previously described
ndez et al. in Arnetoli et al. (2008) and slightly modified by Ferna (2014). HPLC separation was carried out in a Water 296 and the resulting fractions detected at 210 nm with a Waters 2996 photodiode-array detector. The quantitative and qualitative analyses of the chromatograms obtained were made by comparison of retention times and peak areas at 210 nm against standards for oxalic, tartaric, malic, malonic, citric, succinic and glutaric acids (Sigma Aldrich). 2.7. Statistical analysis To evaluate the effect of mycorrhization and the type of soil on the measured variables a two-way Analysis of variance (ANOVA) was used. Log transformation was applied to approximate normality when necessary. When F ratio was significant (p  0.05), Tukey's least significant difference test (HSD, p  0.05) was employed to compare between individual means. A principal component analysis (PCA) was performed using the logtransformed data of metal accumulation of the different substrates. Results are expressed as the mean ± standard error of at least three independent replicates. Data were analyzed using R (version 3.3.1, http://www.r-project.org/) with the packages mixOmics (for PCA, version 6.0.1, http://www.mixOmics.org) and agricolae (version 1.2e4, http://tarwi.lamolina.edu.pe/~fmendiburu). 3. Results 3.1. Soil analysis The physicochemical parameters of the polluted soils are summarized in Table 1. Terronal soil, due to its texture dominated by sand particles (more than 85% of total), was classified as loamy sand with moderately alkaline pH (around 8.7) following the United States Department of Agriculture (USDA) specifications. Nitrastur soil (60e70% of sand particles) was classified as sandy loam with a moderately acidic pH (5.7). Both soils had organic matter contents

3

pH 6.22 ± 0.35 Org. matter (%) 75.30 Texture (%) Sand 65.21 Silt 34.14 Clay 0.72 Total metal (mg kg1) Fe 1530 ± 113 Mn nd Cu 10.20 ± 0.97 Zn 14.19 ± 1.19 As 6.93 ± 0.91 Cd nd Hg nd Pb 17.06 ± 1.15 Available metal (mg kg1) Fe 454.3 ± 23.8 Mn nd Cu 4.31 ± 0.23 Zn 8.61 ± 0.35 As 1.09 ± 0.11 Cd nd Hg nd Pb 2.13 ± 0.20

Terronal

Nitrastur

8.74 ± 0.47 2.54

5.71 ± 0.32 2.87

84.78 14.97 0.26

65.85 33.62 0.52

181065 ± 28302 4175 ± 505 24.47 ± 1.50 519.7 ± 19.4 29.42 ± 2.04 0.83 ± 0.09 1.87 ± 0.31 189.1 ± 35.7

333439 ± 54640 157.3 ± 17.8 1175 ± 160 1848 ± 230 1859 ± 219 7.08 ± 0.92 1.70 ± 0.23 13204 ± 1550

1298 ± 112 550.1 ± 63.5 14.63 ± 1.82 297.1 ± 22.3 17.64 ± 0.91 0.66 ± 0.02 0.68 ± 0.16 81.29 ± 1.2

2391 ± 77 11.80 ± 0.7 115.9 ± 5.9 109.9 ± 12.9 101.4 ± 3.6 0.41 ± 0.06 0.17 ± 0.08 1528 ± 111

between 2e3%. The analysis of total metal concentration of the soil showed high concentrations of Pb, Zn, Cu and As in Nitrastur soil, while Cd and Hg appeared in much lower concentrations. Except for Mn, metal concentrations in Terronal soil were several times lower than in Nitrastur soil. There were also large differences in metal bioavailability between both soils (Table 1). In Nitrastur soil most of the metal was found in non-available fractions (especially in the residual fraction, data not shown), ranging from 79% of the total Pb to 99% of the Fe. On the contrary, Terronal soil showed a high metal bioavailability for all metals except Fe and Mn, ranging from 43% to 80% of the total for Pb and Cd, respectively. Therefore, the bioavailable concentrations of Mn, Zn, Cd, and Hg in this soil were higher than in Nitrastur. Both total and bioavailable metal concentrations of the control substrate (sphagnum peat:perlite mixture) were very low, even below the detection limit in many cases. 3.2. Plant growth and biomass After 60 days of culture, no significant differences in shoot length were detected between M and NM plants cultured in the different soils (Fig. 1). Mycorrhization did not increase the root length in plants grown in the same soil, and even in plants grown in Terronal, root length was reduced (Fig. 1). When compared with the plants cultured in the non-polluted soil, root length was reduced in M plants but not in the NM ones (Fig. 1). As regards biomass production, M plants produced more biomass (measured as dry weight) than the NM ones when they were grown in the control substrate and in Nitrastur soil but not in Terronal soil (Fig. 2). 3.3. Metal accumulation The highest metal accumulation was generally found in plants grown in Nitrastur soil (Table 2) and it was higher in roots in both polluted soils for all metals except for Mn and Zn. By contrast, leaves were generally the main metal-storage organ in plants


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Fig. 1. Shoot and root length of mycorrhizal (M) and non-mycorrhizal (NM) B. pubescens grown for 60 days in control, Terronal and Nitrastur soil. Different letters (upper case for comparison within shoot lengths and lower case for comparisons within root lengths) denote significant differences on HSD test at p < 0.05.

Fig. 2. Fresh weight (FW) and dry weight (DW) of mycorrhizal (M) and non-mycorrhizal (NM) B. pubescens grown for 60 days in control, Terronal and Nitrastur soil. Different letters (upper case for comparison within FW and lower case for comparisons within DW) denote significant differences on HSD test at p < 0.05.

grown in the control substrate. The effect of mycorrhization on metal accumulation depended on the type of soil considered. Thus, M plants cultured in nonpolluted soil showed higher accumulation of Mn and Hg in leaves and Fe, Mn, Cu and Hg in roots; but lower Pb in leaves and As in both leaves and roots (Table 2). In Terronal soil, mycorrhization

slightly increased Mn, Cu, Cd, Hg and Pb leaf accumulation but, except for Mn, had almost no effect in their roots (Table 2). However, when plants were cultured in the most polluted soil (Nitrastur), mycorrhization strongly reduced metal accumulation, except for Mn in leaves and Fe in roots, where no significant differences were observed (Table 2).

Table 2 Metal accumulation in leaves and roots of mycorrhizal (M) and non-mycorrhizal (NM) B. pubescens grown for 60 days in control, Terronal or Nitrastur soil. Different letters within the same column and organ denote significant differences on HSD test at p < 0.05. Organ

Leaves

Treatment

Control Terronal Nitrastur

Roots

Control Terronal Nitrastur

Metal concentration (mg kg

1

DW)

Fe

Mn

NM M NM M NM M

76.45 ± 4.71 d 57.69 ± 2.18 d 288.6 ± 24.2 c 367.4 ± 52.1 c 3046 ± 83 a 661.8 ± 109.7 b

45.87 64.55 79.54 146.2 698.0 565.6

NM M NM M NM M

49.42 ± 0.13 d 77.08 ± 2.18 c 2985 ± 178 b 3809 ± 531 b 17813 ± 2609 a 12729 ± 707 a

3.68 ± 0.01 d 4.95 ± 0.15 c 56.11 ± 9.12 b 86.42 ± 24.29 a 74.29 ± 1.60 a 57.32 ± 2.57 b

± ± ± ± ± ±

0.61 6.87 2.45 22.9 68.5 29.8

d c c b a a

Cu

Zn

As

Cd

2.25 ± 0.14 e 2.38 ± 0.22 e 4.49 ± 0.17 d 5.71 ± 0.62 c 16.71 ± 0.91 a 7.63 ± 0.57 b

112.9 ± 6.8 c 112.8 ± 0.7 c 109.9 ± 3.2 c 124.9 ± 11.9 c 1228 ± 149 a 929.9 ± 27.5 b

0.53 ± 0.08 d 0.33 ± 0.03 e 1.53 ± 0.05 c 1.95 ± 0.24 c 21.62 ± 4.89 a 12.05 ± 1.24 b

0.24 0.20 0.10 0.15 9.45 6.34

Hg

4.43 ± 0.01 e 9.28 ± 2.11 d 17.79 ± 4.83 c 22.17 ± 6.81 c 567.7 ± 59.9 a 364.6 ± 30.6 b

43.44 ± 0.34 c 54.71 ± 13.08 c 47.84 ± 7.39 c 52.89 ± 13.54 c 1208 ± 177 a 803.9 ± 81.2 b

0.08 ± 0.01 d 0.004 ± 0.000 e 8.35 ± 0.25 c 9.92 ± 0.87 c 281.2 ± 43.5 a 196.5 ± 22.4 b

0.12 ± 0.00 e 0.14 ± 0.00 de 0.18 ± 0.01 cd 0.18 ± 0.01 c 22.99 ± 3.28 a 12.07 ± 0.95 b

± ± ± ± ± ±

0.01 0.01 0.01 0.02 0.83 0.64

c c e d a b

0.01 0.02 0.08 0.11 0.08 0.01

Pb ± ± ± ± ± ±

0.00 0.00 0.02 0.02 0.00 0.00

c b a a a bc

0.001 ± 0.000 e 0.01 ± 0.00 d 0.27 ± 0.03 c 0.34 ± 0.03 c 1.43 ± 0.14 a 0.87 ± 0.08 b

0.50 ± 0.03 e 0.29 ± 0.01 f 0.86 ± 0.03 d 1.17 ± 0.18 c 140.1 ± 4.2 a 48.85 ± 2.86 b 0.002 ± 0.000 d 0.002 ± 0.000 d 5.65 ± 0.29 c 6.61 ± 0.93 c 2239 ± 230 a 1450 ± 147 b


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When the fraction of metal bound to the cell wall was analyzed, we observed that, except for As, more than 76% of the metal accumulated in leaves of NM plants was bound to the cell wall in both polluted soils (Fig. 3). Cadmium and Hg concentrations in the soluble fraction were under the detection limits (Fig. 3). In roots, more than 95% of metal detected was bound to the cell wall, and so metal concentration in the soluble fraction was minimal (Fig. 3). It should be noted that NM plants grown in Terronal soil, whose total metal accumulation was significantly lower than that of plants grown in Nitrastur soil (Table 2), had a higher percentage of As and Pb total concentration in the soluble fraction. Mycorrhization did not have significant influence over the percentage of metal bound to the cell wall in either leaves or roots. (Fig. 3). A principal components analysis was performed using the total accumulation data, allowing us to identify the correlations between the eight heavy metals simultaneously. The results showed that samples from the same soil formed well-defined groups (Fig. 4A and B). In both leaves and roots, component 1 explained most of the variation (more than a 90% of the total variation), and showed the samples from left to right in order of increasing metalaccumulation (Fig. 4A and B). According to this component 1, leaf samples from control and Terronal soil, especially the NM ones, almost grouped together (Fig. 4A). On the other hand, component 2 explained very little variation (only 6% of the total variation). In leaves, component 2 was mainly related to Hg accumulation (Fig. 4A), while in roots it seemed to discriminate between plants grown in acidic (Control and Nitrastur) and alkaline soils (Terronal) (Fig. 4B). It is worth noting the clear discrimination between M and NM leaves samples grown in Nitrastur (Fig. 4A). 3.4. Analysis of non-protein thiols After 60 days of culture, changes in the concentration of NPTs in roots and leaves of B. pubescens M and NM were observed among

5

the treatments (Table 3). Interestingly, in all the treatments the concentration of NPTs was always much higher in leaves than in roots. In leaves of M and NM plants grown in control substrate we detected five NPTs, including GSH, PC4, low quantities of PC5, desGly-PC2, and desGly-PC3 (Table 3). Plants cultured in Terronal soil, both M and NM, presented higher levels of GSH than the controls, although no significant differences were observed in their total NPTs concentration (Table 3). Plants grown in Nitrastur soil showed the highest NPTs concentration, mainly due to an increased production of GSH and PC4 and de novo synthesis of PC2 and PC3 (Table 3). Mycorrhization reduced the concentration of NPTs in leaves of plants grown in Nitrastur soil, mainly through a reduction in the concentration of PC4 and PC5 (Table 3). In roots of control plants only GSH, PC4 and PC5 were observed (Table 3). De novo synthesis of NPTs was detected when M and NM plants were grown in Terronal (PC3) and Nitrastur soil (PC3 and especially free cysteine) (Table 3). However, despite the synthesis of new thiol compounds, the total NPTs concentration of NM plants did not differ from those of control plants, since they were counteracted by reductions in the levels of PC4 (Terronal and Nitratur) and GSH (Nitrastur soil). The effect of mycorrhization over the levels of NPTs depended on the type of soil, reducing them in control plants, due to a GSH reduction, and increasing it in those grown in Terronal (Table 3). No differences were observed between M and NM plant grown in Nitrastur soil. 3.5. Analysis of organic acids The analysis of leaf extracts showed the presence of tartaric, malic, malonic and citric acids (Table 4). Except for citric acid, the same compounds were also detected in root extracts (Table 4). The total OA concentration was always higher in leaves than in roots. In leaves, NM control plants presented the highest total OAs

Fig. 3. Percentage of metal in the soluble fraction and bound to the cell wall in leaves and roots of mycorrhizal (M) and non-mycorrhizal (NM) B. pubescens grown for 60 days in Terronal and Nitrastur soil.


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Fig. 4. Biplots of the principal component analysis calculated with the accumulation of each element in leaves (A) and roots (B) of mycorrhizal (M) and non-mycorrhizal (NM) B. pubescens grown for 60 days in control, Terronal and Nitrastur soil.

Table 3 Non-protein thiols (NPTs) concentration in leaves and roots of mycorrhizal (M) and non-mycorrhizal (NM) B. pubescens grown for 60 days in control, Terronal or Nitrastur soil. Different letters within the same column and organ denote significant differences on HSD test at p < 0.05. Nd ¼ not detected. Organ

Treatment

nmol GSH g1 FW Cys

GSH

Leaves

Control

NM M Terronal NM M Nitrastur NM M

nd nd nd nd nd nd

15.17 15.45 19.18 20.77 19.47 18.31

Roots

Control

nd 3.77 nd 3.12 nd 4.12 nd 5.36 2.36 ± 0.05 a 2.23 2.36 ± 0.20 a 2.45

NM M Terronal NM M Nitrastur NM M

desGly-PC2

PC2 ± ± ± ± ± ±

± ± ± ± ± ±

0.50 0.41 0.83 0.45 1.10 0.55

0.16 0.14 0.13 0.01 0.25 0.17

b b a a a a

b c b a d d

nd 0.60 nd 0.81 nd 0.78 nd 1.00 1.77 ± 0.13 a 0.69 1.52 ± 0.06 a 0.85 nd nd nd nd nd nd

± ± ± ± ± ±

0.05 0.16 0.06 0.06 0.07 0.07

nd nd nd nd nd nd

a a a a a a

PC3

desGly-PC3

nd nd nd nd 0.64 ± 0.06 a 0.85 ± 0.17 a

1.32 1.45 1.05 0.99 1.54 1.00

nd nd 0.11 0.21 0.43 0.46

± ± ± ± ± ±

0.21 0.20 0.04 0.02 0.15 0.09

a a a a a a

nd nd ± 0.04 b nd ± 0.09 b nd ± 0.06 a nd ± 0.03 a nd

PC4

desGly-PC4

Total NPTs

15.36 ± 1.08 bc 16.6 ± 0.98 b 13.55 ± 0.53 bc 10.41 ± 0.49 c 22.45 ± 1.12 a 17.18 ± 0.10 b

2.58 ± 0.26 ab 2.72 ± 0.23 a 3.34 ± 0.25 a 2.5 ± 0.08 ab 2.88 ± 0.12 a 1.98 ± 0.10 b

36.17 ± 1.66 c 38.48 ± 1.71 c 39.97 ± 1.16 bc 37.2 ± 2.14 c 50.6 ± 2.13 a 42.32 ± 0.76 b

0.81 0.63 0.28 0.36 0.23 0.27

± ± ± ± ± ±

0.10 0.13 0.01 0.01 0.03 0.02

a ab bc bc c c

0.23 0.19 0.28 0.21 0.32 0.32

± ± ± ± ± ±

0.05 0.04 0.01 0.01 0.04 0.03

a a a a a a

5.16 4.13 5.01 6.42 5.89 6.26

± ± ± ± ± ±

0.17 0.30 0.17 0.11 0.33 0.27

b c bc a ab a

total OA levels in control plants (due to a reduction in the concentration of malic acid) and increased it in those grown in Nitrastur soil (Table 4). In roots, mycorrhization reduced the levels of OAs in control

concentration, which was reduced in plants grown in metalpolluted soils (Table 4). This reduction was especially important in the case of malic acid, both in plants grown in Terronal (90% reduction) and Nitrastur soil (37%). Mycorrhization reduced the

Table 4 Organic acids (OAs) concentration in leaves and roots of mycorrhizal (M) and non-mycorrhizal (NM) B. pubescens grown for 60 days in control, Terronal or Nitrastur soil. Different letters within the same column and organ denote significant differences on HSD test at p < 0.05. Nd ¼ not detected. Organ

Treatment

Leaves

Control

mmol g1 FW Tartaric

Terronal Nitrastur Roots

Control Terronal Nitrastur

Malic

Malonic

Citric

NM M NM M NM M

1.81 1.87 1.44 1.35 1.69 1.82

± ± ± ± ± ±

0.42 0.46 0.19 0.15 0.24 0.17

a a a a a a

11.56 ± 0.42 a 8.86 ± 0.91 b 1.64 ± 0.06 c 1.35 ± 0.10 c 7.29 ± 0.44 b 8.62 ± 0.16 b

12.36 ± 0.04 a 10.92 ± 0.39 ab 13.52 ± 0.91 a 12.53 ± 0.51 a 8.22 ± 0.36 c 9.57 ± 0.17 bc

0.94 0.93 1.56 1.62 0.17 0.17

NM M NM M NM M

0.28 0.23 0.53 0.46 0.22 0.19

± ± ± ± ± ±

0.03 0.02 0.05 0.03 0.01 0.03

b c a a c c

0.63 0.48 0.39 0.34 0.52 0.44

± ± ± ± ± ±

0.01 0.02 0.05 0.02 0.05 0.03

a bc bc c ab bc

0.68 0.52 0.82 0.82 0.56 0.48

± ± ± ± ± ±

0.01 0.03 0.05 0.01 0.06 0.03

b c a a c c

nd nd nd nd nd nd

± ± ± ± ± ±

Total OAs 0.20 0.29 0.15 0.17 0.05 0.03

a a a a b b

26.67 22.57 17.87 16.23 17.37 20.17 1.61 1.22 1.75 1.62 1.30 1.15

± ± ± ± ± ±

± ± ± ± ± ±

0.53 0.51 1.08 0.21 0.50 0.40

0.04 0.04 0.05 0.05 0.11 0.09

a b d d d c

a b a a b b


ndez-Fuego, D., et al., Metal accumulation and detoxification mechanisms in mycorrhizal Betula Please cite this article in press as: Ferna pubescens, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.07.072


ndez-Fuego et al. / Environmental Pollution xxx (2017) 1e10 D. Ferna

plants but had no effect in plants grown in both metal-polluted soils (Table 4). Quantitative changes in the concentrations of some OAs in roots of NM plants were observed when comparing between control and metal-polluted soils (Table 4). Thus, plants grown in Terronal soil presented the highest levels of tartaric and malonic acids and those grown in Nitrastur soil the lowest. Additionally, plants grown in Nitrastur soil showed a significant reduction in their total OA concentration in comparison with plants grown in control or Terronal soil (Table 4). 4. Discussion The relevance of the physicochemical parameters of the soils in the establishment and maintenance of mycorrhizal associations are well known (Carrenho et al., 2007). In our case, both polluted soils had sandy textures, characterized for their porosity and good aeration. This type of soils stimulate mycorrhiza colonization and development, since their low fertility increases the dependence of plants on mycorrhizal associations (Carrenho et al., 2007). The total concentrations of heavy metals in Nitrastur soil were high for several elements. The contents of Zn, Cu, Pb, and As were about 6, 8, 44, and 93 times higher, respectively, than the maximum threshold permitted for an agricultural soil (Kabata-Pendias, 2010). Despite the high concentration of metals found in this soil, the sequential fractionation analysis (BCR method) showed that more than 90% of the Fe, Cu, Zn, As, Cd, and Hg was present in the nonavailable fractions, although the concentration of metal in available fractions was high enough to require soil remediation (KabataPendias, 2010). In these available fractions, metals can be directly absorbed by roots or become available under several chemical or biological processes (Li et al., 2010). Lead was the element with the highest bioavailable concentration and taking into account its high toxicity, this element, along with As, represents the highest environmental risk in Nitrastur soil. Metal concentrations in Terronal soil were also high: Mn, Zn, As, and Hg also exceeded the maximum permitted for an agricultural soil (Kabata-Pendias, 2010), although in a lower magnitude than in Nitrastur. The results of growth and biomass production (Figs. 1 and 2) showed the great capacity of B. pubescens to adapt and tolerate the presence of metals in the soil, since neither growth inhibition nor biomass reduction were observed over the culture period. The ability of different birch species to tolerate the presence of heavy metals, even at high concentrations, has also been reported for
ndez et al., 2008; Bojarczuk and Kieliszewskaother authors (Ferna Rokicka, 2010). The metal tolerance allows birches to act as pioneer species in degraded environments, and to be one of the few woody plants capable to survive in highly-polluted industrial sites (Kozlov, 2005). It has been stated that metal-tolerant birches have a reduced metal translocation to their aboveground parts as a mechanism that allows them to survive in heavy-metal polluted soils (Kopponen et al., 2001). Our data support this hypothesis, although Zn, and specially Mn, showed a high root-to-shoot translocation rate. The high Zn translocation in birch, in comparison with other woody species, has also been reported by other authors although the reason is still not clearly understood (Dmuchowski et al., 2012). The high Zn and Mn translocation observed could be related to the function of these elements in plant cells, since they play an essential role in redox reactions and as enzyme cofactors in many metabolic pathways (Taiz et al., 2015). It is also known that they are actively absorbed by specific membrane transporters (Pilon et al., 2009; Luo et al., 2014) while other heavy metals, such as Cd, As, or Pb, without a known function in the cells, are only taken up due to their chemical similarity to other essential elements like Ca, P, Mg, Fe or Zn (Luo et al., 2014).

7

Once roots absorb the metals, our results confirm that the cell wall is the first barrier to avoid metal toxicity in B. pubescens and hence it constitutes the main sink for heavy metals. Our results also suggest that the importance of the cell wall in metal detoxification increases proportionally with metal accumulation, being of special importance in roots, in which the highest accumulation was detected. Similar results have also been reported in other plant
ndez et al., 2014), suggesting that species (Weng et al., 2012; Ferna root cell walls are the main structures sequestering metals in the plant and the reason for the low root-to-shoot metal translocation observed. Even though for some metal the potentially available fraction was apparently higher in Terronal soil, the highest metal accumulation was generally found in plants grown in Nitrastur soil. Metal availability from soil to plants is known to be strongly dependent €ssler et al., 2010; Bolan on the pH (Sukreeyapongse et al., 2002; Fa et al., 2014). Based on our results, it can be speculated that the moderately alkaline pH of Terronal soil reduced metal bioavailability and therefore limited the uptake of metals by the plants. The correction of soil pH by using soil amendments, such as citric acid, elemental sulphur or other compounds, could be a strategy to consider for enhancing the phytoremediation potential for this soil (Gao et al., 2010; Wiszniewska et al., 2016). Nevertheless, metal mobilizing strategies should be carefully applied since they can also enhance the leaching of metal to the groundwater and therefore increase the environmental risk (Bolan et al., 2014). It is known that mycorrhization increases the root absorption area of trees and therefore the uptake of important nutrients (Luo et al., 2014), which enhances tree fitness and improves their growth in mineral deficient or degraded soils, such as heavy metal polluted soils (Adriaensen et al., 2006; Colpaert et al., 2011; Mrnka et al., 2012). In our case, the positive effect of mycorrhization was clear in control substrate, where it increased the absorption of nutritional elements (e.g. Fe, Mn and Cu) involved in different biological processes (Taiz et al., 2015) and boosted biomass production. In the moderately polluted soil (Terronal), M plants showed increased accumulation of essential metals (e.g. Mn and Cu) in their leaves, but also non-essential ones (e.g. Cd, Hg or Pb) when compared to NM plants. This enhanced accumulation of nonessential elements had no negative effect on plant biomass despite the reduction in root length. By contrast, in the highly polluted soil (Nitrastur), mycorrhization significantly reduced metal accumulation, which lead to an increased biomass when compared to NM plants. Bojarczuk and Kieliszewska-Rokicka (2010) also reported a protecting role of mycorrhiza on birch seedlings by restricting the allocation of Cu and Pb to the leaves. Similar results were also reported in other plant species (Khan et al., 2000; Krznaric et al., 2009, 2010; Colpaert et al., 2011). According to our results, the effect of the mycorrhization of birch with P. ammoniavirescens was apparently related to the concentration of metal in the soil, enhancing metal uptake at low concentrations (control and Terronal soil) and reducing it at high metal concentrations (Nitrastur soil). This response led to an enhanced growth and biomass production of the host plant when growing in the most polluted soil. However, the possibility that this response could be due to an unmeasured soil factor (such us an organic pollutant) affecting plant growth should not also be discarded. Regarding the concentration of NPTs, we observed that most of the NPTs detected in plants grown in polluted soils were also present in the control, which supports the hypothesis that, in plant cells, NPTs may also play a role in the maintenance of metal ion homeostasis (Vurro et al., 2011; Akhter et al., 2012; Fern
andez et al., 2014). As discussed above, plants grown in Nitrastur soil presented the highest metal accumulation, so it should be expected to find the


ndez-Fuego, D., et al., Metal accumulation and detoxification mechanisms in mycorrhizal Betula Please cite this article in press as: Ferna pubescens, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.07.072

8


ndez-Fuego et al. / Environmental Pollution xxx (2017) 1e10 D. Ferna

highest NPTs concentration in them, as our results confirmed. Additionally, they also presented the highest levels of de novo synthesized NPTs. The absence of de novo synthesis of NPTs in leaves of plants grown in the moderately polluted soil (Terronal) could be explained by the fact that these plants were able to maintain a low concentration of metals in their leaves and so their constitutive NPTs levels may be high enough to cope with this metal excess. Nevertheless, it is remarkable that NPTs concentration in roots was always lower than in leaves, despite the higher metal accumulation in this organ (except for Zn and Mn). In previous experiments, we had observed that when plants of B. pubescens and its closely related species B. celtiberica were cultured in vitro or hydroponically, they presented higher NPTs concentration in roots, which was also the organ with the highest metal accumulation (Mesa et al., 2017). These results were in line with those reported for other plant species (Son et al., 2012;
ndez et al., 2014). Although we do not have a clear explanaFerna tion for this apparent contradiction, according to our observations in birch, one might speculate that it can be in part result of the different type of samples. Thus, the roots of birches from pot experiments are usually older and have more secondary growth than those grown in vitro (personal observation). Therefore, they present a higher proportion of lignified and suberized cells, which are less physiologically active (Ye, 2002). Nevertheless, further analysis should be necessary to verify this hypothesis. Plants grown in both polluted soils presented higher concentration of GSH in their leaves, which highlights the importance of this thiol in metal detoxification. Besides being a precursor of PCs synthesis, GSH also plays a role in metal chelation, the control of gene transcription, and the maintenance of the cellular redox state (Jozefczak et al., 2012; Zagorchev et al., 2013). The roots of plants grown in both polluted soils presented synthesis of new thiols, especially those grown in Nitrastur, in which we detected PC3 and Cys accumulation. Cysteine is the main sulfur donor in the biosynthesis of sulfur rich compounds such as methionine, GSH or stress-related proteins (Na and Salt, 2011; Zagorchev et al., 2013). Therefore, the accumulation of Cys observed in plants grown in Nitrastur could be related either to the chelating response or to the biosynthesis of more GSH that could be used, for instance, in the synthesis of new NPTs (PC3). Nevertheless, it is difficult to justify that the birch tolerance to the high concentration of heavy metals accumulated in its tissues could only be result of the differences observed in the concentration of NPTs. We therefore suggest that,
ndez as proposed by other authors (Zagorchev et al., 2013; Ferna et al., 2014), the synthesis of NPTs, due to its high energy cost, would not be the preferred mechanism for long-term metal detoxification in plants exposed to chronic metal-stress. There is some controversy in the role of OAs in heavy metals
ndez et al., 2014). In tolerance in plants (Sun et al., 2011, 2013; Ferna our case, a reduction in the concentration OAs was observed in leaves of plants grown in both polluted soils. A similar trend was also observed in roots, although only in plants grown in Nitrastur. Similar results were reported by Iori et al. (2012), concluding that the observed OAs reduction could be due to the high metabolic cost of coping with the metal-induced oxidative stress. Supporting this hypothesis, we observed increased OAs levels in M plants grown in Nitrastur soil, whose metal accumulation was lower than in the NM ones. On the other hand, plants grown in Terronal soil showed higher levels of tartaric and malonic acids in their roots, which suggests a potential chelating role of these OAs in roots in correlation with a high metal accumulation in this organ. Nevertheless, in plants under acute metal stress, as the plants grown in Nitrastur, this mechanism seems to be replaced by a greater sequestration of metal to the cell wall. Mycorrhization had a variable effect on the levels of both NPTs

and OAs, which was directly related to its impact on metal accumulation. Thus, when comparing M and NM plants, we observed that mycorrhization reduced metal accumulation and therefore NPTs concentration in leaves of plants grown in Nitrastur soil, and increased them in roots of plants grown in Terronal. These changes may be of significant importance for the plant. For instance, given the high-energy cost associated to the synthesis of NPTs, the reduction observed in leaves of M plants grown in Nitrastur soil may allow these plants to save critical energy that could be used in other processes e.g. plant growth, as previously discussed (Fig. 2). Differences between M and NM plants were also observed in plants grown in control substrate. In these plants, a reduction of the concentration of OAs was observed in leaves and roots of mycorrhizal plants. It is known that in the mycorrhizal symbiosis the fungus receives organic compounds from the host plant (Johansson et al., 2008). Therefore, the mentioned reduction could be the result of the fungus’ need for OAs. 5. Conclusions The effect of mycorrhization of B. pubescens with P. ammoniavirescens on metal accumulation appeared to be affected by the concentration of metal in the soil, enhancing metal uptake at low concentrations and reducing it at high metal concentrations. This response led to an enhanced growth and biomass production of the host plant when growing in the highly metal-polluted soil. While NPTs reduced heavy metals toxicity once they got inside the cells, OAs did not seem to play a major role in metal detoxification. Nevertheless, the high metal accumulation observed in birch plants was hardly explained exclusively by the changes in the concentration of NPTs or OAs observed. Thus, we suggest that the sequestration to the cell wall is the main detoxification strategy in white birch exposed to acute chronic metal-stress. We can also conclude that according to the high root-to-shoot translocation rates observed, Betula pubescens is a very promising species for the phytoremediation of soils polluted with Mn and Zn. Acknowledgments This research was supported by the projects CTM2011-29972
ndez-Fuego D. was funded by and LIFE11/ENV/ES/000547. Ferna fellowship FICYT (BP11-140). The authors are grateful to E. Rodrí
s (Environmental technology, biotechnology and guez-Valde geochemistry group, University of Oviedo, Mieres) for his help with soil characterization. References Adriaensen, K., Vangronsveld, J., Colpaert, J.V., 2006. Zinc-tolerant Suillus bovinus improves growth of Zn-exposed Pinus sylvestris seedlings. Mycorrhiza 16, 553e558. http://dx.doi.org/10.1007/s00572-006-0072-7. Akhter, M.F., McGarvey, B., Macfie, S.M., 2012. Reduced translocation of cadmium from roots is associated with increased production of phytochelatins and their precursors. J. Plant Physiol. 169, 1821e1829. http://dx.doi.org/10.1016/ j.jplph.2012.07.011. Arnetoli, M., Montegrossi, G., Buccianti, A., Gonnelli, C., 2008. Determination of organic acids in plants of Silene paradoxa L. by HPLC. J. Agric. Food Chem. 789e795. http://dx.doi.org/10.1021/jf072203d. Bojarczuk, K., Kieliszewska-Rokicka, B., 2010. Effect of ectomycorrhiza on Cu and Pb accumulation in leaves and roots of silver birch (Betula pendula Roth.) seedlings grown in metal-contaminated soil. Water. Air. Soil Pollut. 207, 227e240. http:// dx.doi.org/10.1007/s11270-009-0131-8. Bolan, N., Kunhikrishnan, A., Thangarajan, R., Kumpiene, J., Park, J., Makino, T., Kirkham, M.B., Scheckel, K., 2014. Remediation of heavy metal(loid)s contaminated soils - to mobilize or to immobilize? J. Hazard. Mater 266, 141e166. http://dx.doi.org/10.1016/j.jhazmat.2013.12.018. Bonet, A., Lelu-Walter, M.-A., Faugeron, C., Gloaguen, V., Saladin, G., 2016. Physiological responses of the hybrid larch (Larix  eurolepis Henry) to cadmium exposure and distribution of cadmium in plantlets. Environ. Sci. Pollut. Res. 23, 8617e8626. http://dx.doi.org/10.1007/s11356-016-6094-6.


ndez-Fuego, D., et al., Metal accumulation and detoxification mechanisms in mycorrhizal Betula Please cite this article in press as: Ferna pubescens, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.07.072


ndez-Fuego et al. / Environmental Pollution xxx (2017) 1e10 D. Ferna Carrenho, R., Farto, S., Trufem, B., Lúcia, V., Bononi, R., Silva, E.S., 2007. The effect of different soil properties on arbuscular mycorrhizal colonization of peanuts, sorghum and maize. Acta Bot. Bras. 21, 723e730. http://dx.doi.org/10.1590/ S0102-33062007000300018. Chen, G., Liu, Y., Wang, R., Zhang, J., Owens, G., 2013. Cadmium adsorption by willow root: the role of cell walls and their subfractions. Environ. Sci. Pollut. Res. Int. 20, 5665e5672. http://dx.doi.org/10.1007/s11356-013-1506-3. Cobbett, C., Goldsbrough, P., 2002. Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. http:// dx.doi.org/10.1146/annurev.arplant.53.100301.135154. Colpaert, J.V., Wevers, J.H.L., Krznaric, E., Adriaensen, K., 2011. How metal-tolerant ecotypes of ectomycorrhizal fungi protect plants from heavy metal pollution. Ann. For. Sci. 68, 17e24. http://dx.doi.org/10.1007/s13595-010-0003-9. Corso, G.D., Borgato, L., Furini, A., 2005. In vitro plant regeneration of the heavy metal tolerant and hyperaccumulator Arabidopsis halleri (Brassicaceae). Plant Cell. Tissue Organ Cult. 82, 267e270. http://dx.doi.org/10.1007/s11240-0051314-7. DalCorso, G., Manara, A., Furini, A., 2013. An overview of heavy metal challenge in plants: from roots to shoots. Metallomics 5, 1117. http://dx.doi.org/10.1039/ c3mt00038a. Dmuchowski, W., Baczewska, A.H., Gozdowski, D., 2012. Silver birch (Betula pendula Roth) as zinc hyperaccumulator. In: Nriagu, J., Pacyna, J., Szefer, P., Markert, B., Wuenschmann, S., Namiesnik, J. (Eds.), Heavy Metals in the Environment. Maralte BV, Voorschoten, The Netherlands, pp. 19e25. http://dx.doi.org/ 10.5645/b.2.2. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70e77. http://dx.doi.org/10.1016/0003-9861(59)90090-6. €ssler, E., Robinson, B.H., Stauffer, W., Gupta, S.K., Papritz, A., Schulin, R., 2010. Fa Phytomanagement of metal-contaminated agricultural land using sunflower, maize and tobacco. Agric. Ecosyst. Environ. 136, 49e58. http://dx.doi.org/ 10.1016/j.agee.2009.11.007.
ndez, R., Bertrand, A., Casares, A., García, R., Gonza
lez, A., Tame
s, R.S., 2008. Ferna Cadmium accumulation and its effect on the in vitro growth of woody fleabane and mycorrhized white birch. Environ. Pollut. 152, 522e529. http://dx.doi.org/ 10.1016/j.envpol.2007.07.011.
ndez, R., Bertrand, A., García, J.I., Tame
s, R.S., Gonza
lez, A., 2012. Lead accuFerna mulation and synthesis of non-protein thiolic peptides in selected clones of Melilotus alba and Melilotus officinalis. Environ. Exp. Bot. 78, 18e24. http:// dx.doi.org/10.1016/j.envexpbot.2011.12.016.
ndez, R., Ferna
ndez-Fuego, D., Bertrand, A., Gonza
lez, A., 2014. Strategies for Ferna Cd accumulation in Dittrichia viscosa (L.) Greuter: role of the cell wall, nonprotein thiols and organic acids. Plant Physiol. Biochem. 78, 63e70. http:// dx.doi.org/10.1016/j.plaphy.2014.02.021. Gao, Y., Miao, C., Mao, L., Zhou, P., Jin, Z., Shi, W., 2010. Improvement of phytoextraction and antioxidative defense in Solanum nigrum L. under cadmium stress by application of cadmium-resistant strain and citric acid. J. Hazard. Mater 181, 771e777. http://dx.doi.org/10.1016/j.jhazmat.2010.05.080. Iori, V., Pietrini, F., Massacci, A., Zacchini, M., 2012. Induction of metal binding compounds and antioxidative defence in callus cultures of two black poplar (P. nigra) clones with different tolerance to cadmium. Plant Cell. Tissue Organ Cult. 108, 17e26. http://dx.doi.org/10.1007/s11240-011-0006-8. ISO 1320, 2009. Particle Size Analysis d Laser Diffraction Methods. Jentschke, G., Goldbold, D.L., 2000. Metal toxicity and ectomycorrhizas. Physiol. Plant 109, 107e116. http://dx.doi.org/10.1034/j.1399-3054.2000.100201.x. Johansson, E.M., Fransson, P.M.A., Finlay, R.D., Van Hees, P.A.W., 2008. Quantitative analysis of root and ectomycorrhizal exudates as a response to Pb, Cd and as stress. Plant Soil 313, 39e54. http://dx.doi.org/10.1007/s11104-008-9678-1. Jozefczak, M., Remans, T., Vangronsveld, J., Cuypers, A., 2012. Glutathione is a key player in metal-induced oxidative stress defenses. Int. J. Mol. Sci. 13, 3145e3175. http://dx.doi.org/10.3390/ijms13033145. Kabata-Pendias, A., 2010. Trace Elements in Soils and Plants. CRC Press, Boca Raton, Florida. http://dx.doi.org/10.1201/b10158-25. Khan, A.G., Kuek, C., Chaudhry, T.M., Khoo, C.S., Hayes, W.J., 2000. Role of plants, mycorrhizae and phytochelators in heavy metal contaminated land remediation. Chemosphere 41, 197e207. http://dx.doi.org/10.1016/S0045-6535(99) 00412-9. Kopponen, P., Utriainen, M., Lukkari, K., Suntioinen, S., K€ arenlampi, L., €renlampi, S., 2001. Clonal differences in copper and zinc tolerance of birch in Ka metal-supplemented soils. Environ. Pollut. 112, 89e97. http://dx.doi.org/ 10.1016/S0269-7491(00)00096-8. Kozlov, M.V., 2005. Pollution resistance of mountain birch, Betula pubescens subsp. czerepanovii, near the copper-nickel smelter: natural selection or phenotypic acclimation? Chemosphere 59, 189e197. http://dx.doi.org/10.1016/j.chem osphere.2004.11.010. Krzesłowska, M., 2011. The cell wall in plant cell response to trace metals: polysaccharide remodeling and its role in defense strategy. Acta Physiol. Plant. http://dx.doi.org/10.1007/s11738-010-0581-z. Krznaric, E., Verbruggen, N., Wevers, J.H.L., Carleer, R., Vangronsveld, J., Colpaert, J.V., 2009. Cd-tolerant Suillus luteus: a fungal insurance for pines exposed to Cd. Environ. Pollut. 157, 1581e1588. http://dx.doi.org/10.1016/ j.envpol.2008.12.030. Krznaric, E., Wevers, J.H.L., Cloquet, C., Vangronsveld, J., Vanhaecke, F., Colpaert, J.V., 2010. Zn pollution counteracts Cd toxicity in metal-tolerant ectomycorrhizal fungi and their host plant, Pinus sylvestris. Environ. Microbiol. 12, 2133e2141. http://dx.doi.org/10.1111/j.1462-2920.2009.02082.x.

9

Li, J., Lu, Y., Shim, H., Deng, X., Lian, J., Jia, Z., Li, J.J., 2010. Use of the BCR sequential extraction procedure for the study of metal availability to plants. J. Environ. Monit. 12, 466e471. http://dx.doi.org/10.1039/b916389a. Luo, Z.-B., Wu, C., Zhang, C., Li, H., Lipka, U., Polle, A., 2014. The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot. 108, 47e62. http://dx.doi.org/10.1016/j.envexpbot.2013.10.018.
ndez, D., Walker, D.J., Romero-Espinar, P., Flores, P., del Río, J.A., 2011. Martínez-Ferna Physiological responses of Bituminaria bituminosa to heavy metals. J. Plant Physiol. 168, 2206e2211. http://dx.doi.org/10.1016/j.jplph.2011.08.008.
lez-Gil, R., Gonza
lez, A., Weyens, N., Lauga, B., Mesa, V., Navazas, A., Gonza
nchez, J., Pela
ez, A.I., 2017. Use of endophytic and rhizosphere Gallego, J.L.R., Sa bateria to improve phytoremediation of arsenic-contaminated industrial soil by authoctonous Betula celtiberica. Appl. Environ. Microbiol. http://dx.doi.org/ 10.1128/AEM.03411-16.
r, M., Cieslarova
, Z., Matejka, P., Sza
kova
, J., Tlustos, P., Vos
Mrnka, L., Kucha atka, M., 2012. Effects of endo- and ectomycorrhizal fungi on physiological parameters and heavy metals accumulation of two species from the family Salicaceae. Water. Air. Soil Pollut. 223, 399e410. http://dx.doi.org/10.1007/s11270-0110868-8. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant 15, 473e497. http://dx.doi.org/ 10.1111/j.1399-3054.1962.tb08052.x. Na, G., Salt, D.E., 2011. The role of sulfur assimilation and sulfur-containing compounds in trace element homeostasis in plants. Environ. Exp. Bot. 72, 18e25. http://dx.doi.org/10.1016/j.envexpbot.2010.04.004. Pilon, M., Cohu, C.M., Ravet, K., Abdel-Ghany, S.E., Gaymard, F., 2009. Essential transition metal homeostasis in plants. Curr. Opin. Plant Biol. 12, 347e357. http://dx.doi.org/10.1016/j.pbi.2009.04.011.
pez-S
Rauret, G., Lo anchez, J.F., Sahuquillo, A., Rubio, R., Davidson, C., Ure, A., Quevauviller, P., 1999. Improvement of the BCR three step sequential extraction procedure prior to the certification of new sediment and soil reference materials. J. Environ. Monit. 1, 57e61. http://dx.doi.org/10.1039/ a807854h.
ndez-Aguado, J.M., Afif, E., Carrero, M., Gallego, J.R., 2011. Feasibility Sierra, C., Mene study on the use of soil washing to remediate the As-Hg contamination at an ancient mining and metallurgy area. J. Hazard. Mater 196, 93e100. http:// dx.doi.org/10.1016/j.jhazmat.2011.08.080. Son, K.H., Kim, D.Y., Koo, N., Kim, K.R., Kim, J.G., Owens, G., 2012. Detoxification through phytochelatin synthesis in Oenothera odorata exposed to Cd solutions. Environ. Exp. Bot. 75, 9e15. http://dx.doi.org/10.1016/j.envexpbot.2011.08.011. Sukreeyapongse, O., Holm, P.E., Strobel, B.W., Panichsakpatana, S., Magid, J., Hansen, H.C.B., 2002. pH-dependent release of cadmium, copper, and lead from natural and sludge-amended soils. J. Environ. Qual. 31, 1901e1909. http:// dx.doi.org/10.2134/jeq2002.1901. Sun, J., Cui, J., Luo, C., Gao, L., Chen, Y., Shen, Z., 2013. Contribution of cell walls, nonprotein thiols, and organic acids to cadmium resistance in two cabbage varieties. Arch. Environ. Contam. Toxicol. 64, 243e252. http://dx.doi.org/ 10.1007/s00244-012-9824-x. Sun, R., Zhou, Q., Wei, S., 2011. Cadmium accumulation in relation to organic acids and nonprotein thiols in leaves of the recently found Cd hyperaccumulator Rorippa globosa and the cd-accumulating plant Rorippa islandica. J. Plant Growth Regul. 30, 83e91. http://dx.doi.org/10.1007/s00344-0109176-6. Sungur, A., Soylak, M., Ozcan, H., 2014. Investigation of heavy metal mobility and availability by the BCR sequential extraction procedure: relationship between soil properties and heavy metals availability. Chem. Speciat. Bioavailab. 26, 219e230. http://dx.doi.org/10.3184/095422914X14147781158674. Taiz, L., Zeiger, E., Moller, I.M., Murphy, A., 2015. Plant Physiology and Development, sixth ed. Sinauer Associates, Inc.
zquez, S., Goldsbrough, P., Carpena, R.O., 2009. Comparative analysis of the Va contribution of phytochelatins to cadmium and arsenic tolerance in soybean and white lupin. Plant Physiol. Biochem. 47, 63e67. http://dx.doi.org/10.1016/ j.plaphy.2008.09.010. Vurro, E., Ruotolo, R., Ottonello, S., Elviri, L., Maffini, M., Falasca, G., Zanella, L., Altamura, M.M., Sanit a di Toppi, L., 2011. Phytochelatins govern zinc/copper homeostasis and cadmium detoxification in Cuscuta campestris parasitizing Daucus carota. Environ. Exp. Bot. 72, 26e33. http://dx.doi.org/10.1016/ j.envexpbot.2010.04.017. Weng, B., Xie, X., Weiss, D.J., Liu, J., Lu, H., Yan, C., 2012. Kandelia obovata (S., L.) Yong tolerance mechanisms to cadmium: subcellular distribution, chemical forms and thiol pools. Mar. Pollut. Bull. 64, 2453e2460. http://dx.doi.org/10.1016/ j.marpolbul.2012.07.047.
Wiszniewska, A., Hanus-Fajerska, E., MuszyNska, E., Ciarkowska, K., 2016. Natural organic amendments for improved phytoremediation of polluted soils: a review of recent progress. Pedosphere 26, 1e12. http://dx.doi.org/10.1016/S10020160(15)60017-0.
jcik, M., Sko
rzyn
ska-Polit, E., Tukiendorf, A., 2006. Organic acids accumulation Wo and antioxidant enzyme activities in Thlaspi caerulescens under Zn and Cd stress. Plant Growth Regul. 48, 145e155. http://dx.doi.org/10.1007/s10725-0055816-4. Ye, Z.-H., 2002. Vascular tissue differentiation and pattern formation in plants. Annu. Rev. Plant Biol. 53, 183e202. http://dx.doi.org/10.1146/annurev. arplant.53.100301.135245. Zagorchev, L., Seal, C.E., Kranner, I., Odjakova, M., 2013. A central role for thiols in plant tolerance to abiotic stress. Int. J. Mol. Sci. 14, 7405e7432. http://dx.doi.org/


ndez-Fuego, D., et al., Metal accumulation and detoxification mechanisms in mycorrhizal Betula Please cite this article in press as: Ferna pubescens, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.07.072

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ndez-Fuego et al. / Environmental Pollution xxx (2017) 1e10 D. Ferna

10.3390/ijms14047405. Zhang, Z.C., Chen, B.X., Qiu, B.S., 2010. Phytochelatin synthesis plays a similar role in shoots of the cadmium hyperaccumulator Sedum alfredii as in non-resistant plants. Plant, Cell Environ. 33, 1248e1255. http://dx.doi.org/10.1111/j.13653040.2010.02144.x.


zquez, S., Esteban, E., Fern
Zornoza, P., Va andez-Pascual, M., Carpena, R., 2002. Cadmium-stress in modulated white lupin: strategies to avoid toxicity. Plant Physiol. Biochem. 40, 1003e1009. http://dx.doi.org/10.1016/S0981-9428(02) 01464-X.


ndez-Fuego, D., et al., Metal accumulation and detoxification mechanisms in mycorrhizal Betula Please cite this article in press as: Ferna pubescens, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.07.072