ARTICLE IN PRESS

G Model
EEB-2698; No. of Pages 16

Environmental and Experimental Botany xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

The role of ectomycorrhizas in heavy metal stress tolerance of host

plants
Zhi-Bin Luo a,b,∗ , Chenhan Wu a , Chao Zhang b , Hong Li c , Ulrike Lipka d , Andrea Polle d
a
College of Forestry and State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, PR China
b
College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, PR China
c
College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, PR China
d
Büsgen-Institut, Department of Forest Botany and Tree Physiology, Georg-August Universität, Büsgenweg 2, 37077 Göttingen, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Ectomycorrhizas (EMs) are mutualistic associations between certain soil fungi and higher plants. EMs can
Received 3 October 2013 modulate the cellular, physiological and molecular processes of host plants, resulting in altered responses
Received in revised form 18 October 2013 of the colonized plants to heavy metals. Progress in elucidating the role of EMs in modulating heavy metal
Accepted 23 October 2013
tolerance of host trees is reviewed. In the last decade, a number of ectomycorrhizal fungal isolates and host
plants have been characterized for their tolerance to heavy metals. Additionally, the cellular processes
Keywords:
have been investigated with regard to heavy metal uptake, transport, distribution, toxicity and detox-
Abiotic and biotic stress
ification by ectomycorrhizal fungi and/or host plants. At the cellular level, mechanisms of heavy metal
Mycorrhizal fungi
Nutrient
detoxification include (i) binding of heavy metals to cell wall and extracellular exudates, (ii) decreased
Reactive oxygen species uptake and/or pumping metal ions out of cytosol, (iii) chelation of metal ions in cytosol, (iv) compart-
Subcellular localization mentation of metals in vacuoles or other subcellular structures, and (v) repair of damaged biomolecules.
Transcriptional regulation The efficiency of these protective measures is often increased by EMs, resulting in improved physiological
status and rescued growth. While physiological and cellular responses to heavy metals have been well
studied, experimental data on the underlying molecular mechanisms, especially those induced by the
interaction of ectomycorrhizal fungi and hosts, are scattered. Progress in genome sequencing of EM part-
ners has revealed the importance of metal transporters in mediating tolerance. A better understanding
of the molecular mechanisms is essential for effective application of selected fungal isolates and hosts to
improve the efficiency of bioremediation on heavy metal polluted sites.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
1.1. Function and ecological significance of ectomycorrhizas
The ectomycorrhiza (EM) is an association formed between
ectomycorrhizal fungi (EMF) and the root tips of many plant species
(Smith and Read, 2008). Most EMF belong to the basidiomycota or
ascomycota and it has been estimated that as many as 10 000 fun-
gal species can form EMs with host plants (Finlay, 2008). The hosts
of EMF are mainly tree species in the families Pinaceae, Fagaceae,
Dipterocarpaceae and Caesalpinoidaceae, distributed in tropic, sub-
tropical, temperate and boreal forests (Smith and Read, 2008).
In functional EMs, fungal hyphae ensheath the root tips to form
the hyphal mantle; inside the mantle at the root surface hyphae
∗ Corresponding author at: Key Laboratory of Environment and Ecology in West-
ern China of Ministry of Education, College of Forestry, Northwest A&F University,
Yangling, Shaanxi, 712100, PR Chin. Fax: +86 29 87092262.
E-mail address: luozbbill@163.com (Z.-B. Luo).
penetrate into the cell wall and grow between epidermal cells and
cortical cells, but never enter the cell lumen of roots (Bonfante and
Genre, 2010). These hyphae form the Hartig net, the interface for
exchange of water, nutrients, and other compounds between both
partners (Nehls et al., 2010). Outside the fungal mantle, hyphae
extend into the forest soil and have access to nutrients unavail-
able to plant roots. Thus, the ectomycorrhizal plants obtain water
and nutrients via the fungal hyphae from the soil, while the fungi
are rewarded with photosynthates or stored carbohydrates of the
host plants (Druebert et al., 2009; Kaiser et al., 2010; Pena et al.,
2010). In forests and plantations, the roots of a distinct tree species
are colonized by many different EMF (Buee et al., 2005; Danielsen
et al., 2012, 2013; Lang et al., 2010, 2013). Different trees species
can be interconnected by the fungal hyphae which form a common
ectomycorrhizal network (Bingham and Simard, 2012b; Martin
et al., 2007; Martin and Nehls, 2009; Rooney et al., 2009). With the
help of this common ectomycorrhizal network, exchange of water
and nutrients between different individual tree can occur, which
may increase the stability and fitness of forest ecosystems against
adverse environments (Bingham and Simard, 2011, 2012a).

0098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.envexpbot.2013.10.018

Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16
2 Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
Trees are long-lived perennial plants and, therefore are likely
to face abiotic and biotic stresses including nutrient deficiency,
drought, salinity, heavy metals and pests during their life time
(Campioli et al., 2012; Chen and Polle, 2010; Osakabe et al., 2011).
For instance, EMs mediate improved drought tolerance of their
host plants (Beniwal et al., 2010, 2011; Luo et al., 2009b; Pena
et al., 2013; Pena and Polle, 2013). EMs can furthermore attenu-
ate salt stress on host plants (Langenfeld-Heyser et al., 2007; Li
et al., 2012; Luo et al., 2009a, 2011). Positive effects of EMs on
host resistance to pests and/or pathogens have been unraveled
(Gange et al., 2005; Hartley and Gange, 2009; Nerg et al., 2008,
2009; Pritsch et al., 2009). EMs have positive effects on plant sur-
vival, even in the absence of a functional symbiosis (Müller et al.,
2013). Recent reviews have focused on mycorrhizal modulation of
host drought and pathogen responses (Hartley and Gange, 2009;
Lehto and Zwiazek, 2011; Muller et al., 2007b). Progress has also
been reviewed in the field of mycorrhiza-modulated host plant
tolerance to heavy metals (Colpaert et al., 2011; Rajkumar et al.,
2012). However, there is rapid progress in the area of the cellular,
physiological and molecular mechanisms underlying the function
of EMs and ectomycorrhizal influence on plant heavy metal toler-
ance. Therefore, we will focus this review mainly on these aspects
of the biotic (EMF) and abiotic (heavy metals) interaction of plants.
1.2. Sources and toxicity of heavy metals
Heavy metals are elements with a density higher than 5 g cm−3 ,
mainly including transition metals, some metalloids, lanthanides,
and actinides (Schützendübel and Polle, 2002). Not all of these
heavy metals are biologically important. Among these metals, some
elements such as Zn, Cu and Mn, are required as micronutrients, but
higher concentrations of these heavy metals can be toxic (Ducic
et al., 2006; Ducic and Polle, 2007; Langer et al., 2009). Other heavy
metals such as Cd, As (a metalloid, but hereon also included as a
heavy metal), Pb, Hg and Ni, are non-nutritional and toxic elements
for most organisms (He et al., 2011, 2013a; Jourand et al., 2010;
Krupa and Kozdroj, 2007).
Heavy metals occur naturally in soil with large variations in their
concentrations. Notably, they are steadily accumulating in the envi-
ronment because of the rapid expansion of industrial activities and
their non-degradability (Clemens et al., 2013). Because of their tox-
icity, the enrichment of heavy metals, particularly of the ions of
heavy metals, leads to decreases in biodiversity and productivity
and, thereby, results in changes in structure and function of ecosys-
tems (Mayor et al., 2013; Niemeyer et al., 2012; Pauwels et al.,
2008). Heavy metals in soils can be taken up by plants and accumu-
late in edible parts, which may eventually enter the human body
through the food chain (Kaplan et al., 2011). Some heavy metals
are carcinogenic and toxic, affecting the central nervous system
(i.e., Mn, Hg, Pb, As), the kidneys or liver (i.e., Hg, Pb, Cd, Cu), skin,
bones and teeth (i.e., Ni, Cd, Cu) (Bertin and Averbeck, 2006). Thus,
toxic heavy metals in ecosystems pose a serious threat to human
health.
The typical concentrations (␮g g−1 ) of heavy metals in most
plants are 50 for Zn, 10 for Cu, 200 for Mn, 0.05 for Cd, 0.1 for
As, 1 for Pb and 1.5 for Ni (van der Ent et al., 2013). In con-
trast, plants with foliar concentrations (␮g g−1 ) of heavy metals
above 10,000 for Zn and Mn, 1000 for Cu, As, Pb and Ni and 100
for Cd are regarded as hyperaccumulators for the respective ele-
ment (van der Ent et al., 2013). Thereby, plants in concert with
EMF and other microbes can be used for bioremediation of heavy
metal polluted soils (Rajkumar et al., 2012). For bioremediation, a
particular challenge is to obtain hyperaccumulating plants and/or
micro-organisms including EMF which can readily absorb high
amounts of heavy metals and transport them to the harvestable
parts. In the past, numerous herbaceous hyperaccumulators of
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
heavy metals have been identified, such as Phytolacca acinosa and
Maytenus founieri for Mn (Fernando et al., 2008; Xue et al., 2004),
Solanum nigrum and Sedum alfredii for Zn (Ferraz et al., 2012; Lu
et al., 2013), Commoelina communis and Elsholtzia splendens for Cu
(Jiang et al., 2004; Wang et al., 2004), Arabidopsis halleri and Noc-
caea caerulescens (formerly known as Thlaspi caerulescens) for Cd
(Hanikenne et al., 2008; Kramer, 2010; Milner and Kochian, 2008),
Pityrogramma calomelanos and Pteris vittata for As (Huang et al.,
2011; Visoottiviseth et al., 2002), Microstegium ciliatum, Polygala
umbonata, Spermacoce mauritiana and Hirschfeldia incana for Pb
(Auguy et al., 2013; Rotkittikhun et al., 2006), Sesbania drummondii
and Brassica juncea for Hg (Shiyab et al., 2009; Venkatachalam et al.,
2009), and Alyssum inflatum, Rinorea bengalensis and Pimelea lep-
tospermoides for Ni (Ghasemi et al., 2009; van der Ent et al., 2013).
However, herbaceous plants produce limited amounts of above-
ground biomass which constrains the amount of absorbed metals.
Furthermore, their root system is also less developed than that of
woody species, which limits the access to heavy metals in deeper
soil layers. Therefore, application of fast growing woody plants,
such as Populus and Salix species, which have large biomass, deep
root systems and can sequester intermediate heavy metal concen-
trations in aerial parts, has been proposed for phytoremediation
(He et al., 2013b; Merkle, 2006; Vollenweider et al., 2006; Zhao and
McGrath, 2009). Roots of poplars and willows form EMs with ecto-
mycorrhizal fungi (Baum and Hrynkiewicz, 2005; Baum et al., 2006,
2009; Danielsen et al., 2012, 2013; Krpata et al., 2008, 2009; Mrnka
et al., 2012; Sell et al., 2005) and therefore not only the perfor-
mance of trees on polluted soils but also their associated microbes
are of high importance for the success of bioremediation. In com-
bination with appropriate ectomycorrhizal fungi, trees can play a
key role in bioremediation of heavy metal polluted soils (Aggangan
and Aggangan, 2012; Krpata et al., 2009; Sousa et al., 2012). Readers
interested in general aspects of bioremediation of plant-associated
microbes, are referred to several recent excellent reviews (Clemens
et al., 2013; Kramer, 2010; Milner and Kochian, 2008; Rajkumar
et al., 2012). Here, we wish to highlight that the interaction of
EMF with trees that affects plant tolerance to heavy metals also
has practical applications and that an improved understanding
of the underlying mechanisms of this abiotic × biotic interaction
contributes to developing improved soil amelioration strategies.
Furthermore, we specifically distinguish the effects of nutritional
(Zn, Cu and Mn) and non-nutritional toxic (Cd, Pb, Hg, Ni and As)
heavy metals.
2. Effects of EMs on nutritional heavy metals in plants
2.1. Uptake, transport and distribution of nutritional heavy
metals
As micronutrients Zn, Cu and Mn are needed in minute amounts
by ectomycorrhizal fungi and plants. The nutrients usually occur in
ionic forms in soil solution or are adsorbed to soil particles and
are taken up by specific transporters localized in the plasma mem-
brane and the internal membrane system of fungal and root cells
(Martin et al., 2008; Pilon et al., 2009). Genome sequencing of Lac-
caria bicolor resulted in the discovery of a number of genes encoding
putative transporters for Zn, Cu and Mn ions (Martin et al., 2008).
To date, only few nutritional heavy metal transporters have been
functionally studied in EMF. For instance, in the ectomycorrhizal
fungus Hebeloma cylindrosporum, a gene (HcZnT1) conferring Zn tol-
erance was isolated and functionally characterized (Blaudez and
Chalot, 2011). HcZnT1 was constitutively expressed in cells of H.
cylindrosporum irrespective of the external Zn status and the cell
type (mycelia, sporocarps or ectomycorrhizas) (Blaudez and Chalot,
2011). Further characterization showed that HcZnT1 was probably
located in the endoplasmic reticulum (ER) membranes (Blaudez
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16
Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
and Chalot, 2011) and suggested that HcZnT1 can supply Zn2+ to the
ER and deliver overloaded Zn2+ out of cytosol. Based on the genome
sequence of Tuber melanosporum and on transcriptome analysis,
Bolchi et al. (2011) found that heavy metal transporters involved
in Zn2+ and Cu2+ trafficking exhibited the highest expression lev-
els in ectomycorrhizas, suggesting active translocation of these ions
from fungal hyphae to roots cells. Recently, OmZnT1 encoding a zinc
membrane transporter has been identified in the heavy metal toler-
ant ericoid mycorrhizal isolate Oidiodendron maius Zn and probably
plays a role in Zn transport across the ER membranes (Khouja et al.,
2013). Currently, no studies have reported transporters involved
in Mn trafficking in EMF. With the advent of molecular tools and
genomics information, we expect rapid progress in understanding
the regulation of nutritional heavy metal uptake by fungal trans-
porters. However, to date, data are very limited.
After uptake in ectomycorrhizal fungal cells, nutritional heavy
metal ions can be transported to the Hartig net, translocated out of
fungal cells into root cells, and further transported to other parts of
plants with the help of plant metal transporters (Pilon et al., 2009).
In plants, a number of membrane transporters exist for Zn, Cu and
Mn, which have, however, mostly been studied in non-mycorrhizal
herbaceous plants (Migeon et al., 2010). In the genome sequence
of the ectomycorrhizal tree, Populus trichocarpa, six genes encod-
ing putative metal tolerance proteins (MTPs) were found (Migeon
et al., 2010; Tuskan et al., 2006). Among these proteins, PtMTP1
was functionally characterized as a Zn transporter facilitating the
Zn transfer into the vacuole (Blaudez et al., 2003). PtMTP11.1 and
PtMTP11.2 are involved in Mn transport and probably located in
Golgi apparatus, suggesting that the Golgi apparatus may play a
role in Mn exocytosis in plants (Migeon et al., 2010; Peiter et al.,
2007).
Copper transporters are required for copper acquisition from
the soil or from cellular storage compartments. In the P. trichocarpa
genome, seven genes encoding putative copper transporters were
identified and supported by EST evidence (Migeon et al., 2010).
Zinc–iron permeases (ZIPs) transport zinc and/or other metal
ions from the extracellular space or organellar lumen into the
cytoplasm. Phylogenetic analysis identified 20 putative PtZIPs in
the black poplar genome (Migeon et al., 2010). Analysis of EST
databases and microarray experiments revealed tissue-specific
expression patterns of the PtZIP transcripts in roots, xylem and
leaves (Migeon et al., 2010). Particularly, PtZIP2 and PtZIP10.2 were
highly expressed in the roots of the black poplar, suggesting that
the latter PtZIPs play crucial roles in the uptake of nutritional metals
(Migeon et al., 2010).
Additionally, plants possess natural resistance-associated
macrophage proteins (NRAMPs), which are divalent ion trans-
porters for a range of metal cations including Mn2+ and Zn2+ (Cellier
et al., 1995; Kaiser et al., 2003; Migeon et al., 2010; Pilon et al., 2009;
Xiao et al., 2008). In P. trichocarpa, 9 PtNRAMPs were identified,
among which PtNRAMP3.1 and PtNRAMP3.2 were highly expressed
in poplar roots (Migeon et al., 2010) in agreement with the expres-
sion of their closest homologues in A. thaliana (Lanquar et al., 2005;
Thomine et al., 2003).
Overall, these results indicate that nutritional heavy metal ions
are absorbed and further transported to different compartments
of the host plant. Obviously, plant uptake of these nutrients is not
dependent on EMF. However, ectomycorrhizal fungi can modulate
nutritional heavy metal concentrations in host plants (see below).
Often, the fungal mantle acts as an effective barrier for heavy metals
(Godbold et al., 1998; Jentschke and Goldbold, 2000) because the
heavy metal concentrations in EMs decrease between rhizomorphs
and mantle and from cortical cells to the central cylinder of the
roots. For instance, in ectomycorrhizas of Pinus sylvestris and Suil-
lus luteus collected on a zinc waste site, Zn concentrations declined
from 12,830 ␮g g−1 in rhizomorphs to 2040–3820 ␮g g−1 in the
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
3
hyphal mantle, to 280–675 ␮g g−1 in cortical cells and to 430 ␮g g−1
in vascular tissues (Turnau et al., 2001). Similarly, the Cu concen-
trations decreased from 420 ␮g g−1 in rhizomorphs to less than
17 ␮g g−1 in the hyphal mantle, 32–79 ␮g g−1 in cortical cells and
6 ␮g g−1 in vascular tissues (Turnau et al., 2001). For the Mn concen-
trations, decreases from 490 ␮g g−1 in rhizomorphs, 88–148 ␮g g−1
in the hyphal mantle, 13–26 ␮g g−1 in cortical cells and 14 ␮g g−1
in vascular tissues were reported (Turnau et al., 2001). Apparently,
in the association of P. sylvestris with S. luteus, the fungal mantle
acts as a biological filter for Zn, but in EMs of this host inoculated
with Hebeloma or Inocybe, Zn concentrations in cortical and vascu-
lar tissues were similar to those in the mantle. Therefore, the fungal
species is important with regard to the accumulation or exclusion of
nutritional heavy metals in host plants (Turnau et al., 2002). Further
examples show, however, EMFs do not form constitutive barri-
ers against heavy metal uptake. For example, in Salix × dasyclados
grown on heavy metal contaminated soil increased concentrations
of Zn in the stems and Cu in the roots were found after inocula-
tion with the ectomycorrhizal fungus Paxillus involutus (Baum et al.,
2006). In two varieties of Douglas fir (Pseudotsuga menziesii var.
menziesii and glauca) colonized with Rhizopogon subareolatus, Mn
was enriched in hyphal mantle, Hartig net, cortical cell vacuole and
central cylinder compared to roots of the non-mycorrhizal controls.
Instead of preventing Mn entry, the EM appeared to have facilitated
transport into the root cells (Ducic et al., 2008). The accumulation
of Mn in these plants was linked with phosphorus (P) availabil-
ity (Ducic and Polle, 2007; Ducic et al., 2012). In fact, strong Mn
accumulation can be observed in the xylem of P-starved compared
with P-sufficient plants (Fig. 1). Since EMF influence plant P nutri-
tion (Plassard and Dell, 2010), indirect effects on Mn uptake and
translocation cannot be excluded.
Although the influence of EM on uptake of nutritional heavy
metals is well known and some transporters have been identi-
fied, a clear picture how changes at the molecular level influence
uptake and translocation of the elements and subsequently in the
plants is still missing. The situation is further complicated by the
fact of different species-specific features that become apparent in
plant–fungal interactions. It would therefore be useful to identify
major transporters across different EMF genera and link measure-
ments of transport activities with the subcellular allocation of the
nutritional heavy metals in plants.
2.2. Plant performance in response to excessive concentrations of
nutritional heavy metals
Although Zn, Cu and Mn are essential elements for ectomyc-
orrhizal fungi and host plants, high concentrations of these heavy
metals in soil can be toxic. The threshold concentrations of these
nutrients for the biological requirements vary among different
organisms. As a general reference for the demand of plants, the con-
centrations of these elements in Long-Ashton (LA) and Hoagland
nutrient solutions can be used: 0.2–1000 ␮M for Zn2+ , 0.2–250 ␮M
for Cu2+ and 2.0–1000 ␮M for Mn2+ (Hewitt, 1966; Hoagland and
Arnon, 1950). Higher concentrations than these values are toxic to
most plants studied.
Currently, few studies have addressed the effects of excessive
concentrations of these nutritional heavy metals on ectomycor-
rhizal plants. For example, ectomycorrhizal P. tremula in metallifer-
ous soil with Zn concentrations up to 1960 ␮g g−1 soil accumulated
much higher Zn concentrations in leaves (1890–2740 ␮g g−1 tissue)
than non-ectomycorrhizal plants (Langer et al., 2012). Although
the Zn concentrations in host plants were elevated in this inter-
action, the Zn translocation factor decreased with Zn addition in
ectomycorrhizal plants, suggesting a barrier effect of EMs on Zn
entry into poplar roots under elevated Zn concentrations (Langer
 4

EEB-2698; No. of Pages 16

G Model
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.

Table 1
The ectomycorrhizas mediated nutritional and non-nutritional toxic heavy metal tolerance of host plants.
Microorganisms Plants Metals Concentrations Medium Duration Biomass increment Metal con. in plants Main effects of EMs on host References
(%)a (%)b plants
Nutritional heavy metals
Cenococcum geophilum Populus tremula L. Zn 0–80 mg kg−1 Soil 88 days 200 −4 to 56 Zn translocation to shoots was Langer et al., 2012
inhibited at high Zn levels.
Paxillus involutus Pax1 Paxillus Salix × dasyclados Zn/Cu 762–886 mg kg−1 Soil 6 months 10 to 43 −12 to 21 (Zn) No significant effects on the total Baum et al., 2006
involutus Pax2 (Zn) −33 to 0 (Cu) uptake of Zn and Cu and even
164–192 mg kg−1 (Cu) caused decreased Cd (Pax2) and
Pb (Pax1) contents in the stems.
Glomus spp. Salix spp. Zn 35–80 mg kg−1 Soil 3 month 51 to 100 NA The use of mycorrhizal fungi Kacprzak and Fijalkowski, 2009
only caused less increase of
willow biomass.
Suillus bovinus Pinus sylvestris Zn 3 ␮M–3 mM Perlite 28 days −17 to 29 NA Increased Zn uptake in root and Bucking and Heyser, 1994
needles.
Suillus bovinus Pinus sylvestris Zn 0.1–150 ␮M Sandy soil 9 months NA −44 to −29 Plants colonized by the Adriaensen et al., 2006

ARTICLE IN PRESS
Zn-tolerant isolate grew much
better and remained

Suillus bovinus Pinus sylvestri Zn
Suillus bovinus NP1 Pinus sylvestris Zn
Suillus bovinus P2
Thelephora terrestris
Laccaria laccata
Scleroderma citrinum
Paxillus involutus
Suillus bovinus Pinus sylvestris Cu
Thelephora terrestris
Suillus bovinus Pinus sylvestris Cu
Suillus bovinus Pinus sylvestris Cu
Pisolithus 23-01 Eucalyptus Cu
Pisolithus H6394 urophylla
Scleroderma Acacia aulacocarpa
Astraeus
Rhizopogon Seudotsuga Mn
subareolatus menziesii var.
menziesii and var.
glauca
Pisolithus tinctorius (Pers.) Betula lenta L. Mn
Coker & Couch
Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
physiologically healthier when
exposed to elevated Zn.
0.1–229 ␮M Soil 6 weeks NA NA The Zn-tolerant S. bovinus Adriaensen et al., 2004
genotype was particularly
efficient in protecting pines
from Zn stress.
7–14 mM Sand–vermiculite 4 months −40 to 4 (shoots) −49 to 22 These mycorrhizal fungi act as a Colpaert and Vanassche, 1992
mixture safety net that can immobilize
large amounts of zinc.
0.3–47 ␮M Soil 22 to 36 days 75 −13 to 56 (shoots) Both ectomycorrhizal fungi Van Tichelen et al., 2001
protect P. sylvestris against Cu
toxicity.
6.5–25 mg L−1 Perlite 4 weeks −8 to 42 NA No significant improvement in Huang and Tao, 2004
reducing the physiological
restrain in ectomycorrhizal
seedlings.
0.005–4.8 mM Soil 10 weeks −55 to 43 NA Root growth and nutrient Adriaensen et al., 2005
uptake of plants colonized by
the Cu-tolerant isolate were
hardly affected under Cu stress
0–750 ␮M Soil–sand mixture 3 months −67 to 24 −44 to 303 Seedlings inoculated with Aggangan and Aggangan, 2012
Pisolithus, Scleroderma and
Astraeus grew better than the
non-mycorrhizal ones.
10 mM Peat–sand mixture 5 months NA −18 to 40 Accumulation of high Mn was Ducic et al., 2008
not prevented in inoculated
seedlings.
0.25–500 ␮M Soil 30 months 23 to 104 −33 to 5 Inoculated seedlings had lower Walker et al., 2004
concentrations of potentially
phytotoxic metallic elements,
particularly Mn, than
uninoculated seedlings.

Non-nutritional toxic heavy metals

Suillus luteus Pinus sylvestris Cd 10 mM Perlite 8 weeks NA NA Seedlings inoculated with fungi Krznaric et al., 2010
collected from the polluted site
reduced metal transfer to their
host and maintained nutrient
acquisition under high metal
exposure.
Suillus luteus Pinus sylvestris Cd 0–20 ␮M Perlite 8 weeks NA −67 to 12 Mycorrhizal pines showed Krznaric et al., 2009
better plant protection: more
fungal biomass and a higher
nutrient uptake at high Cd
exposure. Less Cd was
transferred to aboveground
plant parts.
 EEB-2698; No. of Pages 16
G Model
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.

Paecilomyces lilacinus NH1 Solanum nigrum L. Cd 50 mg kg−1 Soil 2 to 10 weeks NA NA Application of CA and PLNH1 Gao et al., 2010
significantly improved the
antioxidative defense in S.
nigrum under Cd stress.
Hebeloma mesophaeum Salix viminalis Cd 7.8 mg kg−1 Soil 3 months NA −2 to 38 Inoculated willows showed Hrynkiewicz et al., 2012
increased concentrations of
nutritional elements and
decreased concentrations of Cd
in the shoots.
Suillus bovinus Pinus pinaster Cd 0–30 mg kg−1 Soil 6 months −94.02 −19.85(shoots) Colonization by S. bovinus Sousa et al., 2012
Rhizopogon roseolus significantly enhanced shoot
development up to 30% in
contaminated soil while

ARTICLE IN PRESS
colonization by R. roseolus
produced no significant effect.

Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
Laccaria Pinus sylvestris L. Cd 20 ␮g ml−1 Soil 3 months NA NA The concentration of cadmium Krupa and Piotrowska-Seget, 2003
laccata (Scop.:Fr), in plants inoculated with fungi
Rhizopogon luteolus (Corda) was significantly lower than in
Th.M, Xerocomus badius non-inoculated seedlings.
Amanita rubescens Pinus sylvestris Cd 10 ␮g ml−1 Soil 10 week NA NA Elevated accumulation of Cd in Kozdroj et al., 2007
Hebeloma sinapizans underground parts of the
Pseudomonas putida inoculated seedlings was found.
Bacillus cereus
ECM(various) Populus tremula Cd 5.7–111 mg kg−1 Soil NA NA NA Aspen trees accumulated Zn and Krpata et al., 2009
Cd to similar concentrations as
the EMF.
Paxillus filamentosus (Scop) Fr. Betula celtiberica 5– 10 mg kg−1 Peat:vermiculite 15 days 46 to 153 −2 to 767 Plants of Fernandez et al., 2008
Dittrichia viscosa (1:10) mycorrhized white
Cd birch grown in the
presence of Cd had
a better
development and
accumulated more
Cd in their shoots
than the
non-mycorrhized
ones.
Paxillus involutus Populus Canadensis 1.91 mg kg−1 Soil 11 weeks 11 to 33 45 to 81 P. involutus Sell et al., 2005
Hebeloma crustuliniforme Salix viminalis significantly
Pisolithus tinctorius Cd enhanced the total
Cd extraction by P.
canadensis.
Boletus edulis Pinus tabulaeformis Cd 0–3 mg kg−1 Soil 12 weeks NA NA The content of exchangeable Cd Huang et al., 2008
in the mycorrhizosphere
decreased remarkably.
Cadophora finlandica Salix caprea Cd 32.7 mg kg−1 Soil 19 weeks NA NA Significantly increased Cd Utmazian et al., 2007
Salix smithiana concentrations in leaves of both
willow species.
Xerocomus chrysenteron Pinus tabulaeformis Cd 0–20 mg L−1 Perlite 6 weeks NA NA While APase activity in exudates Zheng et al., 2009
of fungi and seedlings decreased
under Cu and Cd stress in
comparison to that of the
control, the APase activity in
seedlings was maintained by
inoculation.
Laccaria bicolor (Maire) Orton Picea abies(L.) Cd 0–5 ␮M Sand 9 weeks NA −56 to 54 Paxillus involutus ameliorated Jentschke et al., 1999
S238 Paxillus involutus Karst.) the negative effects of 0.5 M Cd
(Batsch) Fr. 533 on shoot and root growth and
chlorophyll content of old
needles, whereas L. bicolor did
not.

5
 6

EEB-2698; No. of Pages 16

G Model
Table 1 (Continued)
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.

Microorganisms Plants Metals Concentrations Medium Duration Biomass increment Metal con. in plants Main effects of EMs on host References
(%)a (%)b plants
Thelephora terrestris Ehrh. ex Pinus sylvestris Cd 44.5 ␮M Perlite 2 months −45 to 14 (shoot) −74 to −28 A protective effect against Colpaert and Vanassche, 1993
Fr. cadmium toxicity in the host
Laccaria laccata was observed with all
(Scop, ex Fr.) Cooke mycobionts.
Scleroderma citrinum Pers.
Paxillus involutus (Batsch ex
Fr.) Fr., two strains of Suillus
luteiis (L. ex Fr.) S. F. Gray
three isolates of Suillus
bovinus (L. ex Fr.) O. Kuntze.
One isolate of 5. luteus
two isolates of 5. bovinus
Laccaria laccata Picea abies (L.) Karst Cd 190 ␮M Peat–vermiculite 6 days NA −61 (shoot) Cadmium content of needles of Galli et al., 1993
(Scop, ex Fr.) (1:14, v/v) mycorrhizal plants was
significantly lower than that of
non-mycorrhizal plants.
Paxillus involutus Betula pendula Roth Cd 0–200 ␮M Peat–vermiculite 30 min NA NA The mycorrhizal birch seedlings Blaudez et al., 2000

ARTICLE IN PRESS
revealed high efficiency of N

Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
acquisition under metal
exposure might be regarded as a
mechanism of stress avoidance.
Paxillus involutus Pinus sylvestris Cd 0–25 ␮g g−1 Peat:vermiculite 8 weeks NA NA Ectomycorrhizal colonization of Hartley et al., 1999
(1:5, v/v) Scots pine was shown to be
more sensitive than Scots pine
itself to Cd and Zn, but prior
colonization did provide a
competitive advantage with
respect to biomass production.
Paxillus involutus Pinus sylvestris Cd 0–25 ␮g g−1 Peat–vermiculite 8 weeks NA NA P. involutus ameliorated the Hartley-Whitaker et al., 2000
toxicity of Cd to P. sylvestris.
Suillus variegatus Pinus sylvestris Cd 0–100 mg kg−1 Peat–vermiculite 8 months NA NA Colonisation by S. bovinus Kim et al., 2004
Paxillus involutus improved shoot growth,
colonisation by Paxillus involutus
had no effect on growth of
seedlings.
Glomus intraradices Salix alba L. Pb/Cd. NA Soil 6 months NA −33 to −8 H. mesophaeum strongly reduced Mrnka et al., 2012
Hebeloma mesophaeum Populus nigra L. the accumulation of Cd and Fe in
willow and poplar shoots.
Scleroderma citrinum, Amanita Pinus sylvestris L. Pb/Cd 10 ␮gml−1 (Cd) Soil 12 weeks NA −38 to 7 Inoculation of pine seedlings Krupa and Kozdroj, 2007
muscaria Lactarius rufus 40 ␮gml−1 (Pb) significantly reduced
translocation of Zn(II), Cd(II) or
Pb(II) from roots to shoots.
EMF (various) Betula pendula Roth Pb/Cd NA Soil NA NA NA Fivefold higher concentrations Krupa and Kozdroj, 2004
of the metals were found in the
mycorrhizal roots.
Paxillus involutus Pinus sylvestris L. Pb/Cd NA Soil NA NA NA On the polluted substrate the Werner et al., 2004
Laccaria laccata mycorrhizal roots showed
Suillus luteus significantly lower content of
terpenoids than nonmycorrhizal
roots.
Paxillus involutus Picea abies Pb 5 ␮M Sand 32 to 42 days NA NA P. involutus decreased Pb Marschner et al., 1996
Pisolithus tinctorius translocation from the roots to
Lccaria laccata the stems.
EMF (12 morphotypes) Betula pendula Roth. Pb 1459–1812 mg kg−1 Soil 4 months NA NA The addition of unpolluted Bojarczuk and Kieliszewska-Rokicka, 2010
forest soil to the
metal-contaminated soil
resulted in the enrichment of
the soil substrate by propagules
of EMF.
Paxillus involutus Piua abies Pb 5 ␮M Soil 3 weeks NA NA Total Pb in roots was reduced by Jentschke et al., 1998
Laccaria bicolor mycorrhizal infection.
Hebeloma crustuliniforme Pinus sylvestris Pb NA Soil:gravel:sphagnum 5 to 17 months NA NA Inoculation of pine seedlings Werner and Karolewski, 2004
(Bull.) Quél. (strain 22-1) peat (1:1:3, v/v) resulted in a decrease in
Paxillus involutus (Batsch) Fr. concentration of phenolics in
(strain 5-1), Laccaria laccata roots and except for seedlings
(Scop. ex Fr.) (strain 9-1) inoculated with Laccaria laccata
Suillus bovinus (Fr.) Knutze negatively influenced the
(strain 15-4), Suillus luteus above-ground part:root ratio.
(Fr.) S.F.
Gray (strain 14-1)
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16

Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx 7

et al., 2012). Under elevated Zn2+ concentrations, a Zn-tolerant iso-

Metal concentrations in leaves or shoots were calculated as metal concentrations in ectomycorrhizal plants minus metal concentrations in non-ectomycorrhizal plants divided by metal concentrations in non-ectomycorrhizal
Ahonen-Jonnarth and Finlay, 2001
late of the ectomycorrhizal fungus S. bovinus was identified, which

Ahonen-Jonnarth et al., 2000

protected its host, P. sylvestris from Zn toxicity by inhibition of

Jean-Philippe et al., 2011

Zn accumulation in the needles (Adriaensen et al., 2004, 2006).
Johansson et al., 2008

Jourand et al., 2010

Similarly, a Cu-adapted S. luteus isolate provided an effective pro-

Crane et al., 2012

tection against Cu toxicity in seedlings of P. sylvestris exposed to
high Cu concentrations (60 ␮M) and diminished Cu accumulation in

Biomass increment percentage was calculated as biomass of ectomycorrhizal plant minus biomass of non-ectomycorrhizal plant divided by biomass of non-ectomycorrhizal plant multiplied with 100.
needles compared to those of non-mycorrhizal plants (Adriaensen
et al., 2005). In the same line, seedlings of sweet birch (Betula lenta)
inoculated with the ectomycorrhizal fungus Pisolithus tinctorius
Exudation generally increased in

ones and had better P, K, Mg and

contained lower Mn concentrations in leaves under elevated Mn

The mycorrhizal seedlings grew

no protective effects against Hg

mycorrhizal treatments than in

Seedling survival declined with

ultramafic P. albus were able to

shifts in community structure.

composition was altered with

Soil inoculants from EFPC had

(145–175 ␮g g−1 ) than non-mycorrhizal trees (Walker et al., 2004).
better than non-mycorrhizal
the presence of mycorrhizal
seedlings compared to NM

non-mycorrhizal controls.
Levels of oxalic acid were

Ectomycorrhizal species
increasing Hg additions.
An earlier study has demonstrated that two ectomycorrhizal fungi,
tolerate high and toxic
Plants inoculated with
significantly higher in

concentrations of Ni.

S. bovinus and Thelephora terrestris, forming EMs with P. sylvestris

protected their host against Cu toxicity at high Cu concentrations in
the soil (Van Tichelen et al., 2001). These results suggest that metal-
seedlings.

S status.

toxicity.
tolerant EMF can act as an effective biological barrier against metal
toxicity in host plants (Table 1) and may play a role in large-scale
land reclamation at phytotoxic metalliferous sites.
However, not all ectomycorrhizal fungi protect their host plants
−19 to 10 (shoots)

from toxicity imposed by nutritional heavy metals. For instance,

EMs with Thelephora terrestris (Ehrh.) Fr. increased the Zn con-
−98 to 105

centrations in the shoots of P. sylvestris seedlings compared with

NA

NA

NA

NA

non-mycorrhizal plants and also enhanced Zn toxicity (Colpaert

and Vanassche, 1992). Similarly, in Douglas fir (Pseudotsuga men-
ziesii variety glauca) seedlings inoculated with R. subareolatus, the
−70 to 125 (roots)

Mn concentrations in needles were elevated in comparison with

those of non-mycorrhizal plants, aggravating Mn toxicity in the
host (Ducic et al., 2008).
In spite of some variations, most studies indicate that ectomy-
NA

NA

NA

NA

NA

corrhizal trees maintain better growth than non-ectomycorrhizal

trees when exposed to excess concentrations of nutritional heavy
5 months
15 weeks

11 weeks

12 weeks

metals (Table 1). For the magnitude of the positive effect, the extent
28 days
1 week

by which metal accumulation is prevented does not play a deci-

sive role. Therefore, in addition to shielding the plant from excess
Peat–vermiculite–MMN

uptake, EMF may also enhance the internal detoxification. The

studies also indicate that selection of appropriate ectomycorrhizal
Vermiculite–sand
Sand–vermiculite
Peat:vermiculite

fungal species or isolates is important for utilization of EMs as a

substrate

strategy to remedy soils with Zn, Cu and/or Mn pollution. Effects of

(1:4, v/v)

Sand

EMs on metal uptake, translocation and toxicity in host plants must

Soil

be thoroughly investigated before their application in the field on

a large scale.
10–100 ␮M (Pb)

85–170 ␮M (Ni)

0–366 mg g−1
0–60 mg kg−1
1–10 ␮M (Cd)
1–10 ␮M (As)

100 mg kg−1
0.44 ␮M(Cd)
8.9 ␮M (Cd)

3. Effects of EMs on non-nutritional heavy metals in plants

17 ␮M(Ni)

3.1. Uptake, transport and distribution of non-nutritional heavy

metals
Cd/Pb/As

Cd/Ni

Cd/Ni

Since the non-nutritional heavy metals Cd, Pb, Hg, Ni and As

Hg

Hg
Ni

are toxic for most ectomycorrhizal fungi and plants, from an evo-
lutionary point of view, specific transporters for their uptake and
Platanus occidentalis

transport are unlikely to exist in these organisms. Their transport

Eucalyptus globulus
Pinus sylvestris L.

Pinus sylvestris L.

is thus probably afforded by transport systems for essential ele-

Pinus sylvestris

Pinus rigida

ments such as Ca, Mg, Fe, Zn, Cu, Mn or phosphate, which exist in
ectomycorrhizal fungi and plants (Ma et al., 2013; Mendoza-Cozatl
Karst.

et al., 2011; Pilon et al., 2009; Seth et al., 2012). In EMs, the non-
nutritional toxic heavy metals can be taken up by fungal hyphae
plants multiplied with 100.

and are transported to the Hartig net for loading into root cells.
Ectomycorrhizal communities
Hebeloma velutipes, Paxillus

Alternatively to active transport through the fungal cells and the

NA: no data available.
roseolus, Suillus bovinus
byssinum, Rhizopogon

cortical symplast, the elements can diffuse in the apoplast follow-

involutus, Piloderma

Rhizopogon roseolus
Suillus variegatus

Paxillus involutus

ing the concentration gradient. However, this apoplastic pathway is

Suillus variegatus
Laccaria bicolor

Pisolithus albus

EMF (various)

blocked by the Casparian strip in endodermis of roots and requires

active loading into the vascular system. Consequently, the accumu-
lation of heavy metals in roots is often much higher than in other
a

plant tissues.

Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16

8 Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx

Fig. 1. Imaging of the manganese (Mn) distribution in vessels of poplars supplied with limited (a, c: 0.064 ␮M) or sufficent (b, d: 641 ␮M) phosphorus (P). Images were
generated for the Mn-L edge. The images were generated with an energy-dispersive X-ray scanning/transmission electron microscope (STEM) as described elsewhere
(Abo-Ogiala et al., 2013). The original STEM images are shown in c and d, and the Mn mapings were scaled from 42 to 525 units.

Up to now, no studies have identified and functionally char-
acterized specific transporters for Cd2+ , Pb2+ , Hg2+ , Ni2+ , arsenate
or arsenite in ectomycorrhizal fungi. However, in the genomes of
the ectomycorrhizal fungi L. bicolor and T. melanosporum, several
sequences encoding genes for putative heavy metal transporters
were identified (Martin et al., 2008, 2010). Additionally, the ecto-
mycorrhizal fungus Cadophora finlandica grown in either Cd or
Pb containing medium displayed differential expression of sev-
eral genes predicted to encode heavy metal transporters (Gorfer
et al., 2009). In bacteria, various mercury transporters such as MerC,
MerE, MerF, MerH and MerT, whose main function is to modulate
mercuric ion uptake into the cells, have been identified (Mok et al.,
2012; Yamaguchi et al., 2007). Given the diversity of EMF and occur-
rence under a wide range of environmental conditions, it is likely
that much is to be discovered with regard to heavy metal trans-
porters. The current results, though still very limited, suggest that
in future studies efforts should be undertaken to identify and func-
tionally characterize transporters involved in uptake and transport
of toxic heavy metals in ectomycorrhizal fungi.
Similar to the transporters in ectomycorrhizal fungi, little infor-
mation is available about transporters implicated in uptake and
transport of the non-nutritional toxic heavy metal in ectomycor-
rhizal host plants. Recently, seedlings of P. × canescens inoculated
with the ectomycorrhiza-forming strain MAJ of Paxillus involutus
were found to display enhanced Cd2+ uptake into EMs, which was
associated with upregulation of transcript levels of plant genes
encoding several metal transporters, i.e., ZIP2, NRAMP1.1, HMA4
(Ma et al., 2013). It has been speculated that these genes may be
involved in Cd transport.
Other studies have demonstrated that there is no general
responses with regard to EMs mediated uptake of Cd2+ , Pb2+ ,
Hg2+ , Ni2+ , arsenate or arsenite from the soil (Baum et al., 2006;
Jean-Philippe et al., 2011; Johansson et al., 2008; Jourand et al.,
2010; Krpata et al., 2009; Mrnka et al., 2012; Sell et al., 2005;
Sousa et al., 2012). The presence and concentration of other ions
of respective nutrient elements can affect uptake of toxic com-
pounds because the non-nutritional heavy metals compete with
essential elements for uptake by transporters. For example, Cd
affects the translocation of some metals using the Fe-like or Ca-
like pathway in poplar (P. jacquemontiana var. glauca) (Solti et al.,
2011). For Fe-like metals, Cd can share a common translocation
system, but Cd competes for translocation systems with Ca-like
metals (Solti et al., 2011). P. alba × P. tremula var. glandulosa trans-
formed with yeast cadmium factor 1 (ScYCF1), a gene encoding
a transporter that delivers toxic metal(loid)s into the vacuoles
of budding yeast, accumulated increased amounts of Cd and Pb
compared with the wildtype plants (Shim et al., 2013). These
results show that uptake and transport of the non-nutritional
toxic heavy metals can be influenced by biotechnological means,
but may also disturb the absorption of essential nutritional
metals.
It is well known that herbaceous plants take up and transport
Cd2+ , Pb2+ , Hg2+ , Ni2+ , arsenate and/or arsenite from the soil via dif-
ferent types of transporters such as heavy metal ATPases (HMAs),
NRAMPs, ZIPs and ATP-Binding Cassette transporters (ABCs), whose
main functions are to transport essential elements (Migeon et al.,
2010; Park et al., 2012; Takahashi et al., 2011; Ueno et al., 2011).
Although herbaceous plants are unable to form EMs, their heavy

Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16
Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
metal transport systems can provide insights into similar pro-
cesses in ectomycorrhizal host plants. For example, N. caerulescens
is a Cd hyperaccumulator, which possesses uptake systems for
Cd2+ (Milner and Kochian, 2008): TcHMA3 is located in the tono-
plast and transports Cd into the leaf vacuoles (Ueno et al., 2011).
Higher expression of TcHMA3 confers Cd hyperaccumulation in N.
caerulescens (Ueno et al., 2011). Another member of HMAs in N.
caerulescens, TcHMA4, was demonstrated to play a role in metal
tolerance by mediating active efflux of Cd2+ and Pb2+ out of the
cell (Papoyan and Kochian, 2004). Similarly, elevated Cd2+ uptake
and transport in A. halleri depends on higher expression of the
metal pump HMA4 which is ascribed to modified cis-regulatory
sequences and increased copy numbers of the gene (Hanikenne
et al., 2008). Involvement of HMA2 in Cd uptake and transport
was also demonstrated in rice (Takahashi et al., 2012). OsHMA2
was localized at the plasma membrane and mainly expressed
in rice roots. OsHMA2-suppressed rice displayed decreased foliar
Cd concentrations compared with the wildtype, suggesting that
OsHMA2 plays a role in Cd loading to the xylem (Takahashi et al.,
2012).
NRAMP is also implicated in uptake and transport of the non-
nutritional toxic heavy metals in plants. For instance, OsNRAMP1
in rice was likely located at the plasma membrane; its higher
expression in the roots led to increased Cd accumulation in the
shoots, suggesting that OsNRAMP1 plays a key role in Cd uptake
and transport in rice (Takahashi et al., 2011). Using radioactive
Cd-109 and Zn-65, Zhao et al. (2006) suggested that A. halleri
at least partly employs the Zn pathway to absorb and trans-
port Cd2+ , probably involving several ZIPs (Talke et al., 2006).
The Arabidopsis AtABCC1 and AtABCC2 vacuolar transporters are
important for transport of cytosolic phytochelatin-metal (arsenic,
Cd and Hg) complexes into the vacuoles (Park et al., 2012). Inter-
estingly, it was reported that oilseed rape (Brassica napus) and
white lupin (Lupinus albus) possess an active, saturable component
for Hg uptake (Esteban et al., 2013). In future studies, it will be
worthwhile to investigate the orthologues of these transporters in
ectomycorrhiza-forming plants and to find out how these trans-
porters are regulated by EMF in response to the non-nutritional
toxic heavy metals.
After uptake of the toxic elements, they are distributed in
different tissues, cells and subcellular compartments of the ecto-
mycorrhizal fungi and host plants along the transport pathway.
Heavy metals are mainly found in the hyphal mantle, Hartig net,
root, leaf and phloem (Blaudez et al., 2000; Cocozza et al., 2011; He
et al., 2011, 2013a, 2013b; Kieffer et al., 2008; Krupa and Kozdroj,
2004; Turnau et al., 2001, 2002; Vollenweider et al., 2006, 2011). At
the cellular and subcellular levels, the toxic metals are accumulated
in root epidermal cells, trichomes, in the cell walls and in vacuoles
(Cocozza et al., 2011; He et al., 2013a; Turnau et al., 1996, 2001,
2002; Vollenweider et al., 2006, 2011). Elemental microanalysis
coupled with transmission electron microscopy demonstrated
that in Rhizopogon roseolus EMs of P. sylvestris exposed to calamine
dumps rich in Cd and Pb, the heavy metals were mainly located
in the fungal mantle, Hartig net, vacuoles and extracellular spaces
(Turnau et al., 1996). EMs of P. sylvestris formed with S. luteus
contained higher Pb concentrations in the hyphal mantle than in
the extramatrical mycelia, whereas those formed with Hebeloma
or Inocybe spp. exhibited higher Cd deposition in the extramatrical
mycelia compared with in the hyphal mantle (Turnau et al., 2002).
In a recent study, using histochemical staining, transmission
electron microscope in combination with energy-dispersive X-ray
microanalysis, He et al. (2013a) found that Cd in P. × canescens
was mainly located in root epidermal cells, intercellular spaces of
root cortical cells, phloem, leaf veins and trichomes, and vacuoles
of root epidermal cells, cortical cells, companion cells of phloem,
and of mesophyll cells. In leaves of Salix viminalis, Cd was mainly
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
9
allocated to the collenchyma cell walls of the veins (Vollenweider
et al., 2006). In roots of A. halleri and N. caerulescens, Cd was mainly
deposited in the vacuoles and cell walls (Kupper et al., 2000;
Vazquez et al., 1992). In ectomycorrhizal plants, these metals are
also bound to hyphal cell walls, which therefore, act as biological
filters.
3.2. Plant and fungal performance and toxicity symptoms in the
presence of non-nutritional heavy metals
Ectomycorrhizal fungi and host plants possess a basal toler-
ance to Cd, Pb, Hg, Ni and As (Clemens, 2006). The appearance of
toxicity symptoms such as growth retardation mainly depends on
the heavy metal concentration, duration of exposure, and species,
genotype or isolate of the ectomycorrhizal fungus and the plant
(Table 1) (Clemens, 2006; Colpaert et al., 2011; Krpata et al., 2008,
2009; Patra et al., 2004; Rajkumar et al., 2012; Sell et al., 2005). For
both, EMF and trees, genotypes with increased resistance have been
identified (Colpaert et al., 2011; Fomina et al., 2005; Krznaric et al.,
2009). For instance, in a study investigating the toxicity of Cd and
Pb to an ectomycorrhizal fungal species, more than 60% of fungal
isolates displayed a lethal sensitivity to Pb salts (phosphate, car-
bonate, sulphide, tetraoxide), but metal tolerant isolates with good
growth performance on medium containing these metals were also
identified (Fomina et al., 2005).
When the basal tolerance threshold is exceeded after the
exposure to toxic heavy metals, EMF and host plants display
symptoms of injury such as yellowing and shedding leaves in
plants and necrosis and decreased growth for fungi and plants
(Hall, 2002; Ma et al., 2013). At this stage the metabolism is dis-
ordered including defects in mineral metabolism (Hrynkiewicz
et al., 2012; Jamnicka et al., 2007; Ma et al., 2013; Patra et al.,
2004), inhibited gas exchange (Godbold et al., 1998; Jentschke
et al., 1999; Ma et al., 2013; Mrnka et al., 2012) and dam-
age to the DNA (Patra et al., 2004). To counteract heavy metal
toxicity, which is at least partly caused by an induction of
reactive oxygen species (ROS) (Finlay, 2008; He et al., 2011,
2013b; Ma et al., 2013; Ott et al., 2002; Schützendübel and
Polle, 2002), defenses are activated including differential regu-
lation of gene expression (Ma et al., 2013; Majorel et al., 2012)
and increased the biosynthesis of antioxidants (Schützendübel and
Polle, 2002). As fungi lack ascorbate, activation of glutathione and
phytochelatins is particularly important to avoid stress due to ROS
(He et al., 2013a; Hegedus et al., 2007; Ma et al., 2013; Seth et al.,
2012).
Non-nutritional heavy metals are particularly dangerous
because of their mutagenic potential. Hg, Pb and arsenic injure
mitosis by spindle disturbance due to their affinity to thiol groups
(Patra et al., 2004). Mercury induced c-mitosis, i.e. doubling of
the chromosome without proper sorting to the daughter cell,
thereby, leading to polyploid and aneuploid cells, and c-tumors
in plants (Patra et al., 2004). Lead salts caused numerous c-
mitoses, low mitotic activity and inhibited root growth (Patra et al.,
2004). Furthermore, arsenite is a potent co-mutagen (Patra et al.,
2004).
Often, non-nutritional heavy metals influence the uptake and
concentrations of nutritional heavy metals (Krpata et al., 2008;
Krznaric et al., 2009). The toxicity of non-nutritional toxic heavy
metals to ectomycorrhizal fungi and host plants, thus, can result
from (i) the displacement of essential metals such as Mg2+ , Ca2+ ,
Fe2+ and Zn2+ in biomolecules causing molecular dysfunctions, (ii)
binding of metals to thiol groups which leads to inhibited functions
of the target biomolecules and (iii) as the consequence of blocked
thiol groups overproduction of ROS (Hall, 2002; Schützendübel and
Polle, 2002; Sharma and Dietz, 2009).
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16
10 Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
4. Mechanisms of EM-modulated host plant tolerance to
heavy metals
4.1. Cellular mechanisms
The nutritional heavy metals Zn, Cu and Mn are beneficial to
ectomycorrhizal fungi and host plants in the desirable concentra-
tions range. For instance, Zn ions serve as structural components
in transcription factors and act as co-factors in many enzymes
(Broadley et al., 2007). Under normal conditions, Zn is allocated to
the cytosol, chloroplasts, mitochondria and peroxisomes (Broadley
et al., 2007). At higher concentrations, ectomycorrhizal plants can
alter the cellular availability to minimize toxicity of these metals
(Fig. 2). Emerging evidence suggests that the cellular mechanisms
involved in detoxification of excess heavy metals mainly include (i)
the biofilter function of ectomycorrhizal fungi and (ii) subcellular
sequestration of these metals in ectomycorrhizal plants (Bellion
et al., 2006; Colpaert et al., 2011; Hall, 2002). For example, in P.
sylvestris inoculated with the metal-tolerant fungus S. luteus, a sig-
nificant decrease in the concentrations of Zn, Cu and Mn from
the rhizomorph of the fungus to vascular tissues of host plants
demonstrated filter effects of the ectomycorrhizal fungus and pre-
vented excess metal accumulation in plant roots (Turnau et al.,
2001). Other examples show that ectomycorrhizal plants enhance
the uptake of nutritional heavy metals and sequester them in vac-
uoles and vesicles, thus favoring intracellular detoxification (Ducic
et al. (2008). In the case of Mn, EM-facilitated P-uptake probably led
to formation of Mn-P deposits, and thus, improved Mn detoxifica-
tion. These examples show that understanding of the benefits of the
EMF-plant interaction requires knowledge of the cellular utilization
and localization of nutritional heavy metals.
The toxicity of non-nutritional heavy metals was outlined in
Section 3.2. Their detoxification is important to avoid toxicity to
the two interacting organisms, the EMF and the plant. Ectomycor-
rhizal fungi are likely to employ protective mechanisms similar to
those of their host plants (Bellion et al., 2006; Colpaert et al., 2011;
Hall, 2002) including binding to cell walls and sequestration in
the vacuole. Further protective measures are: (i) binding of heavy
metals to extracellular exudates, (ii) decreased uptake and/or
pumping metal ions out of cytosol via transporters located at the
plasma membrane, (iii) chelation of metal ions in cytosol by com-
pounds, such as glutathione, phytochelatins and metallothioneins,
(iv) compartmentation of metals in other subcellular structures to
avoid accumulation of metal ions in cytosol, and (v) repair of metal
damaged biomolecules (Fig. 2) (Colpaert et al., 2011; Gallego et al.,
2012; Hall, 2002; Schützendübel and Polle, 2002; Seth et al., 2012).
In ectomycorrhizal plants, the extramatrical hyphae and mantle
constitute the first biological barrier against toxic metal ions and
can decrease plant exposure by extracellular binding or seques-
tration in the vacuoles of the fungal cells (Colpaert et al., 2011;
Hall, 2002; Schützendübel and Polle, 2002). Higher heavy metal
concentrations were detected in cell walls and vacuoles of hyphal
cells of EMs than cell walls of root cells (Turnau et al., 1996, 2001,
2002). Binding of Cd to the cell wall and accumulation of Cd in vac-
uole were the two main cellular mechanisms for Cd detoxification
of fungus P. involutus (Blaudez et al., 2000). Ectomycorrhizal Scots
pine seedlings showed increased exudation of organic acids, amino
acids, dissolved monosaccharides and dissolved organic carbon in
comparison with those of non-mycorrhizal plants. These exudates
can chelate Cd2+ and Pb2 and therefore, most likely contributed to
detoxification of the heavy metals and improved the performance
of the seedlings (Johansson et al., 2008). In P. × canescens apart
from cell wall and trichome allocation, Cd was mainly found in
vacuoles of roots, mesophyll and the companion cells of phloem,
suggesting that the plant attempts of immobilize Cd along the
transport path (He et al., 2013a). However, large differences exist
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
between different poplar species with regard to their ability to
prevent transport and to detoxify Cd (He et al., 2013b; Polle et al.,
2013; Zacchini et al., 2009).
4.2. Physiological mechanisms
During the establishment and functioning of EMs, numerous
physiological changes occur in roots and even at the whole-
plant level of the host (Beniwal et al., 2011; Ducic et al., 2008;
Gafur et al., 2004; Langenfeld-Heyser et al., 2007; Li et al., 2012;
Luo et al., 2009a). These physiological responses include altered
uptake of nutrients, particularly of phosphorus, improved water
status and increased osmolality, changed photosynthetic rates
and photosynthate supply to roots, varied concentrations of free
amino acids, phytohormones and fatty acids, and shifts in the
ROS balance (Beniwal et al., 2010; Ducic et al., 2009; Gafur et al.,
2004; Langenfeld-Heyser et al., 2007; Luo et al., 2009a, 2009b;
Schützendübel and Polle, 2002). For instance, in P. × canescens col-
onized with P. involutus, EMs resulted in elevated phosphorus
concentrations in roots and leaves (Luo et al., 2009a, 2011), and
increased net influxes of Ca2+ and K+ into the roots (Li et al., 2012).
Higher photosynthetic rates increased the concentrations of solu-
ble sugars and sugar alcohols in ectomycorrhizal plants (Druebert
et al., 2009; Langenfeld-Heyser et al., 2007; Luo et al., 2009a; Pena
et al., 2013). Improved nutrition of the host plants may therefore
contribute to increased host resistance to upcoming abiotic and
biotic stresses including heavy metals (Luo et al., 2009a, 2011; Ma
et al., 2013; Rajkumar et al., 2012).
Currently, only few studies have investigated EM-triggered
physiological changes in host plants exposed to excess nutritional
heavy metals (Ducic et al., 2006) and thus, this area deserves fur-
ther analyses. For the non-nutritional toxic heavy metals, i.e., Cd,
Pb, Hg, Ni and As, modified tolerance of ectomycorrhizal plants to
these metals is also related to EM-induced physiological changes
in host plants (Fig. 2). Recently, Ma et al. (2013) demonstrated
that P. × canescens inoculated with P. involutus displayed larger root
surface area, higher concentrations of P, Ca, Fe and Zn, increased
photosynthetic rates, soluble sugars and starch, altered concentra-
tions of O2 •− and H2 O2 , and elevated concentrations of ascorbate
and glutathione, suggesting improved nutrient and carbohydrate
status, increased detoxification, and defense preparedness in host
plants, which probably contributed to their enhanced Cd tolerance.
Additionally, exudates of ectomycorrhizal fungi, for example
oxalic acid, that can alter toxic heavy metal bioavailability in the
rhizosphere, may also result in modified tolerance of host plants
to these toxic metals (Bellion et al., 2006; Colpaert et al., 2011;
Meharg, 2003). Formic acid, malic acid and succinic acid were
detected in exudates of four ectomycorrhizal fungal isolates grown
with Modified Melin-Norkans medium (Ray and Adholeya, 2009).
Correlations between organic acid exudation and accumulation of
Cd, Pb, Ni and As in hyphae of the EMF suggest that fungal exudates
play a pivotal role in tolerance of EMs to these heavy metals (Ray
and Adholeya, 2009). In addition to fungal exudates, EMs can also
induce changes in exudates of the host plants in the rhizosphere to
modify metal tolerance. The soluble protein and acid phosphatase
activity in root exudates of P. tabulaeformis inoculated with an
ectomycorrhizal fungus, Xerocomus chrysenteron, were enhanced in
comparison with those of non-ectomycorrhizal roots, which may
increase nutrition and improve Cd tolerance of the host plants
(Zheng et al., 2009). Most studies to date indicate that EMs induce
physiological changes that improve the supply with essential nutri-
ents, alter metabolite levels, activate defense pathways and root
exudates of host plants, thereby resulting in modified tolerance
to the toxic heavy metals of the hosts. Obviously, the improved
nutrition is an important factor in EMF-mediated host tolerance. To
increase our understanding of the physiological regulation, further
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16

Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx 11

Fig. 2. A schematic model for EM-mediated host plant tolerance to the nutritional (Zn, Cu and Mn) and non-nutritional toxic (Cd, Pb, Hg, Ni and As) heavy metals. A tangential-
section view of an EM, uptake of the heavy metals and rhizoshperic exudates (panel a). The physiological processes underlying EM-modulated host plant tolerance to the
heavy metals (panel b). The cellular and molecular processes underlying EM-mediated host plant tolerance to the heavy metals (panel c). The cellular processes for heavy
metal detoxification mainly include (i) binding of heavy metals to cell wall and extracellular exudates, (ii) decreased uptake and/or pumping metal ions out of cytosol, (iii)
chelation of metal ions in cytosol, (iv) compartmentation of metals in vacuoles or other subcellular structures, and (v) repair of metal damaged biomolecules. The molecular
processes in mycorrhizal plants mainly include (i) transcriptional regulation of heavy metal ion transporters, (ii) differentially expressed transcriptomes, and (iii) differentially
expressed proteomes in response to the heavy metal exposure. ZIPs: zinc–iron permeases, NRAMPs: natural resistance-associated macrophage proteins, HMAs: heavy metal
ATPases, ABCC1/2: ATP-binding cassette transporter C1/2, MTPs: metal tolerance proteins, MTs: metallothioneins, PCs: phytochelatins.

studies should pay attention to the uptake dynamics of the heavy
metal ions in relation to the changes in EM-mediated changes in
plant nutrition.
4.3. Molecular mechanisms
Coordinated activation of detoxification processes is required
for the successful defense of heavy metal toxicity. Priming of
these pathways in the host plants appears to be an important
benefits provided by EMF (Fig. 2). Ectomycorrhizal plants employ
several molecular pathways to regulate the interaction between
host plants and heavy metals (Fig. 2): first, EMs can modify the
activities of transporters for metal uptake and transport at the
transcript and protein levels (Ma et al., 2013). Second, EMs can
modulate the availability of chelating molecules for binding heavy
metals in cells (Ma et al., 2013; Seth et al., 2012). Third, EMs can
alter the transcriptomes of host plants to mediate defense against
stresses including heavy metals (Flores-Monterroso et al., 2013;
Heller et al., 2008; Luo et al., 2009a).
For the nutritional heavy metals (Zn, Cu and Mn), most studies
were concerned with Zn. In the ectomycorrhizal fungus, H. cylin-
drosporum, the gene HcZnT1 was isolated and characterized for its
roles in Zn homeostasis and detoxification in fungal cells (Blaudez
and Chalot, 2011). An involvement of metal transporters in Zn tol-
erance was also found in two isolates of S. luteus whose contrasting
expression patterns suggested that Zn uptake and transport is reg-
ulated at the transcriptional level in this fungal species (Muller
et al., 2007a). In poplars, the overexpression of PtHMA4 and PtPCS1

Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16

12 Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx

caused differences in tolerance to Zn exposure, and higher Zn accu- the bark, whose toxicity was probably attenuated by defense acti-
mulation was detected in leaves of a PtPCS1-overexpressing line, vation through a differentially regulated transcriptomic network
indicating that HMA4 and PCS1 play roles in Zn uptake and accu- (He et al., 2013a). Mycorrhizal fungal colonization has an influence
mulation in poplars (Adams et al., 2011). In N. caerulescens, NcZNT1, on the differential expression of transcriptomes and/or proteomes
a ZIP family transporter, is a plasma membrane-localized trans- of host plants under stress (Aloui et al., 2011; Heller et al., 2008;
porter for Zn uptake that is regulated at the transcript and protein Lingua et al., 2012; Luo et al., 2009a). In P. × canescens colonized
levels (Milner et al., 2012). In Thlaspi goesingense, the metal tol- with P. involutus, EMs formation activated stress-related genes and
erance protein 1 (TgMTP1) was involved in Zn accumulation via signaling pathways in host, resulting in priming of pathways and
Zn transport into the vacuoles and its expression in the tonoplast conferring enhanced abiotic stress tolerance in the host plants (Luo
led to increased Zn tolerance in A. thaliana (Gustin et al., 2009). et al., 2009a). So far, improved stress tolerance by EM-induced
Another metal tolerance protein, AtMTP11 was found to be a Golgi- priming has been tested for salt stress (Luo et al., 2009a), but it
localized manganese transporter and plants overexpressing MTP11 is highly probable that the EM-modulated stress-related genes can
conferred hyper-tolerance to Mn (Peiter et al., 2007). Therefore, also lead to increased tolerance to other abiotic stresses including
Peiter et al. (2007) suggested that this gene may achieve Mn toler- heavy metals. Taken together, there is progress in the analysis of the
ance by Golgi-mediated exocytosis. In P. involutus, the transcript molecular mechanisms underlying EM-modulated host plant toler-
levels of Pimt1 encoding a metallothionein was induced by Cu ance to heavy metals. Given the diversity of EMF and the variability
(Bellion et al., 2007). Overexpression of Pimt1 in H. cylindrosporum of host defenses (Colpaert et al., 2011; Schützendübel and Polle,
conferred higher Cu tolerance (Bellion et al., 2007). Collectively, 2002), there is now an urgent need for more systematic approaches
these results suggest that the molecular regulation of genes and/or unraveling the molecular basis of EMF-plant interaction to improve
proteins involved in metal uptake, transport and chelation play heavy metal tolerance. We may anticipate that the ongoing fun-
key roles in tolerance to the nutritional heavy metals. There is ini- gal genome sequencing projects (Plett and Martin, 2011) provide a
tial evidence that metal trafficking is affected by ectomycorrhizal useful basis for future research in this direction.
fungi, thereby affecting plant performance, but most studies to date
have addressed transport processes only in the plants and not in
5. Conclusions and outlook
the associations between EMF and their hosts. The regulation and
protection of trees with excess nutritional heavy metals by EMF
Heavy metal contamination in soils is a serious threat to the
deserves further detailed investigations.
health of human beings. To remedy heavy metal polluted soils,
Information on the molecular regulation of uptake, trafficking
heavy metal tolerant ectomycorrhizal fungal isolates and suit-
and detoxification of the non-nutritional toxic heavy metals (Cd,
able host tree species may be employed for bioremediation as an
Pb, Hg, Ni and As) by EMF and their host trees is also relatively
environmentally friendly technology. In the last decades, progress
limited. As described above, the metal concentrations in ecto-
has been made in the identification of heavy metal tolerant
mycorrhizal plants are influenced by EMs, but are not always
species/isolates of ectomycorrhizal fungi and plants. The com-
diminished (Table 1). For instance, the transcript levels of genes
bined analysis of cellular, physiological and molecular responses
involved in metal uptake, transport and detoxification processes,
of trees and associated ectomycorrhizal fungi has shown that dif-
such as ZIP2, NRAMP1.1, PCS, ABCC1, ATM3 and HMA4, were induced
ferent detoxification strategies can be employed such as binding
in P. × canescens colonized with P. involutus, probably resulting in
of the heavy metals outside the living tissue either to chemical
the enhanced Cd uptake in the host (Ma et al., 2013). The Ni-tolerant
groups in the cell wall or by complexation with exuded organic
isolate of Pisolithus albus was found to improve the growth of its
compounds in the rhizosphere. Metal transporters appear to be
host Eucalyptus globulus at toxic nickel concentrations, which has
crucially involved in tolerance, generally by increasing deposition
mainly ascribed to reduced Ni uptake by the roots and lower trans-
of the toxic elements in the vacuole. However, the overall picture
fer from roots to shoots in ectomycorrhizal plants (Jourand et al.,
how plants can safely accumulate heavy metals or detoxify them
2010). Transcriptome analysis under Ni-stress revealed Ni-induced
with the help of EMF remains scattered. A main reason for the slow
genes encoding proteins putatively involved in the modification of
progress is the huge diversity and host specificity of EMF with spe-
cellular components and molecular functions in P. albus (Majorel
cific response patterns and the influence of EMF on many plant
et al., 2012).
processes, in particular on mineral nutrition, which in turn affects
In the ascomycete, T. melanosporum (truffle) transcriptional
the interaction with other elements such as heavy metals. A further
regulation was detected in response to Cd for metal homeostasis-
challenge is to understand how the utilization of nutritional heavy
related genes involved in metal transport, oxidative stress defense
metals can be optimized and the damage due to non-nutritional
and metal detoxification, e.g., TmelMT, a small-size metallothionein
elements minimized since both groups seem to partly share uptake
and TmelPCS, a phytochelatin synthase, suggesting that transcrip-
pathway and occur frequently together on polluted sites. A bet-
tional regulation of metal homeostasis-related genes plays a crucial
ter understanding of the molecular mechanisms is essential to
role in the fungal tolerance to Cd (Bolchi et al., 2011). Additionally,
improve the efficiency of bioremediation on heavy metal polluted
in another EM-forming ascomycete, Cadophora finlandica, Cd expo-
sites. With the accomplishment of the genome sequencing of sev-
sure led to increased transcript levels of genes encoding multidrug
eral ectomycorrhizal fungi including L. bicolor and T. melanosporum
transporters, again highlighting that transcriptional regulation of
(Martin et al., 2008, 2010), and host trees, such as P. trichocarpa,
genes implicated in metal transport may be important for Cd
(Tuskan et al., 2006); Eucalyptus grandis (Genome Project 2010,
tolerance (Gorfer et al., 2009). Moreover, exposure of the ectomyc-
http://www.phytozome.net/eucalyptus), and Picea abies (Nystedt
orrhizal fungus P. involutus to Cd induced biosynthesis of chelating
et al., 2013), it becomes feasible and advantageous to dissect the
molecules such as glutathione, ␥-glutamylcysteine and metallothi-
cellular, physiological and molecular mechanisms underlying EM-
onein in mycelia, suggesting that the chelation of the toxic heavy
modulated host plant tolerance to heavy metals.
metals in the cytosol with these thiols may be of importance for
metal detoxification and tolerance (Courbot et al., 2004).
Differential changes of the proteome and transcriptome in Acknowledgements
response to toxic heavy metals have been analyzed in tree species
(He et al., 2013a; Kieffer et al., 2008). For instance, exposure of This work is supported by the National Natural Science Foun-
P. × canescens to 200 ␮M Cd resulted in strong accumulation of Cd in dation of China (Grant nos. 31070539, 31100481, 31270647),

Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16
Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
the State Key Basic Research Development Program (Grant No.
2012CB416902), the Special Fund for Forest Science and Technol-
ogy Research in the Public Interest (Grant No. 201204210) and the
Fundamental Research Funds for the Central Universities of China
(Grant No. YQ2013005, QN2013013). The work in the laboratory of
AP is supported by the Federal State of Lower Saxony, the Bunde-
sanstalt für Landwirtschaft und Ernährung (BLE), and the German
Science Foundation (SPP1685: Ecosystem nutrition, Po362/22, INST
186/766-1 FUGG).
References
Abo-Ogiala, A., Carsjens, C., Diekmann, H., Fayyaz, P., Herrfurth, C., Feussner,
I., Polle, A., 2013. Temperature-induced lipocalin (TIL) is translocated under
salt stress and protects chloroplasts from ion toxicity. J. Plant Physiol.,
http://dx.doi.org/10.1016/j.jplph.2013.08.003.
Adams, J.P., Adeli, A., Hsu, C.Y., Harkess, R.L., Page, G.P., dePamphilis, C.W., Schultz,
E.B., Yuceer, C., 2011. Poplar maintains zinc homeostasis with heavy metal genes
HMA4 and PCS1. J. Exp. Bot. 62, 3737–3752.
Adriaensen, K., van der Lelie, D., Van Laere, A., Vangronsveld, J., Colpaert, J.V., 2004. A
zinc-adapted fungus protects pines from zinc stress. New Phytol. 161, 549–555.
Adriaensen, K., Vangronsveld, J., Colpaert, J.V., 2006. Zinc-tolerant Suillus bovi-
nus improves growth of Zn-exposed Pinus sylvestris seedlings. Mycorrhiza 16,
553–558.
Adriaensen, K., Vralstad, T., Noben, J.P., Vangronsveld, J., Colpaert, J.V., 2005. Copper-
adapted Suillus luteus, a symbiotic solution for pines colonizing Cu mine spoils.
Appl. Environ. Microbiol. 71, 7279–7284.
Aggangan, N.S., Aggangan, B.J.S., 2012. Selection of ectomycorrhizal fungi and tree
species for rehabilitation of Cu mine tailings in the Philippines. J. Environ. Sci.
Manage. 15, 59–71.
Aloui, A., Recorbet, G., Robert, F., Schoefs, B., Bertrand, M., Henry, C., Gianinazzi-
Pearson, V., Dumas-Gaudot, E., Aschi-Smiti, S., 2011. Arbuscular mycorrhizal
symbiosis elicits shoot proteome changes that are modified during cadmium
stress alleviation in Medicago truncatula. BMC Plant Biol. 11, 75.
Ahonen-Jonnarth, U., Finlay, R.D., 2001. Effects of elevated nickel and cadmium con-
centrations on growth and nutrient uptake of mycorrhizal and non-mycorrhizal
Pinus sylvestris seedlings. Plant Soil 236, 129–138.
Ahonen-Jonnarth, U., Van Hees, P.A.W., Lundstrom, U.S., Finlay, R.D., 2000. Organic
acids produced by mycorrhizal Pinus sylvestris exposed to elevated aluminium
and heavy metal concentrations. New Phytol. 146, 557–567.
Auguy, F., Fahr, M., Moulin, P., Brugel, A., Laplaze, L., El Mzibri, M., Filali-Maltouf, A.,
Doumas, P., Smouni, A., 2013. Lead tolerance and accumulation in Hirschfeldia
incana, a mediterranean Brassicaceae from metalliferous mine spoils. PLoS ONE
8, e61932.
Baum, C., Hrynkiewicz, K.,2005. Adaptation of ectomycorrhizas of Salix caprea in
soils with high heavy metal contents. In: Referate und Posterpraentationen der
Jahrestagung der Deutschen Bodenkundlichen Gesellschaft, Marburg, Germany,
2–9 September 2005. Deutsche Bodenkundliche Gesellschaft, p. 166.
Baum, C., Hrynkiewicz, K., Leinweber, P., Meissner, R., 2006. Heavy-metal
mobilization and uptake by mycorrhizal and nonmycorrhizal willows
(Salix × dasyclados). J. Plant Nutr. Soil Sci.: Z. Pflanzenernahr. Bodenkd. 169,
516–522.
Baum, C., Toljander, Y.K., Eckhardt, K.U., Weih, M., 2009. The significance of host-
fungus combinations in ectomycorrhizal symbioses for the chemical quality of
willow foliage. Plant Soil 323, 213–224.
Bellion, M., Courbot, M., Jacob, C., Blaudez, D., Chalot, M., 2006. Extracellular and
cellular mechanisms sustaining metal tolerance in ectomycorrhizal fungi. FEMS
Microbiol. Lett. 254, 173–181.
Bellion, M., Courbot, M., Jacob, C., Guinet, F., Blaudez, D., Chalot, M., 2007. Metal
induction of a Paxillus involutus metallothionein and its heterologous expression
in Hebeloma cylindrosporum. New Phytol. 174, 151–158.
Beniwal, R.S., Hooda, M.S., Polle, A., 2011. Amelioration of planting stress by soil
amendment with a hydrogel-mycorrhiza mixture for early establishment of
beech (Fagus sylvatica L.) seedlings. Ann. For. Sci. 68, 803–810.
Beniwal, R.S., Langenfeld-Heyser, R., Polle, A., 2010. Ectomycorrhiza and hydrogel
protect hybrid poplar from water deficit and unravel plastic responses of xylem
anatomy. Environ. Exp. Bot. 69, 189–197.
Bertin, G., Averbeck, D., 2006. Cadmium: cellular effects, modifications of
biomolecules, modulation of DNA repair and genotoxic consequences (a review).
Biochimie 88, 1549–1559.
Bingham, M.A., Simard, S.W., 2011. Do mycorrhizal network benefits to survival and
growth of interior Douglas-fir seedlings increase with soil moisture stress? Ecol.
Evol. 1, 306–316.
Bingham, M.A., Simard, S.W., 2012a. Ectomycorrhizal networks of Pseudotsuga men-
ziesii var. glauca trees facilitate establishment of conspecific seedlings under
drought. Ecosystems 15, 188–199.
Bingham, M.A., Simard, S.W., 2012b. Mycorrhizal networks affect ectomycorrhizal
fungal community similarity between conspecific trees and seedlings. Mycor-
rhiza 22, 317–326.
Blaudez, D., Botton, B., Chalot, M., 2000. Cadmium uptake and subcellular compart-
mentation in the ectomycorrhizal fungus Paxillus involutus. Microbiology (UK)
146, 1109–1117.
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
13
Blaudez, D., Chalot, M., 2011. Characterization of the ER-located zinc transporter
ZnT1 and identification of a vesicular zinc storage compartment in Hebeloma
cylindrosporum. Fungal Genet. Biol. 48, 496–503.
Blaudez, D., Kohler, A., Martin, F., Sanders, D., Chalot, M., 2003. Poplar metal tolerance
protein 1 confers zinc tolerance and is an oligomeric vacuolar zinc transporter
with an essential leucine zipper motif. Plant Cell 15, 2911–2928.
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, 227–240.
Bolchi, A., Ruotolo, R., Marchini, G., Vurro, E., di Toppi, L.S., Kohler, A., Tisserant, E.,
Martin, F., Ottonello, S., 2011. Genome-wide inventory of metal homeostasis-
related gene products including a functional phytochelatin synthase in the
hypogeous mycorrhizal fungus Tuber melanosporum. Fungal Genet. Biol. 48,
573–584.
Bonfante, P., Genre, A., 2010. Mechanisms underlying beneficial plant–fungus inter-
actions in mycorrhizal symbiosis. Nat. Commun. 1, 48.
Broadley, M.R., White, P.J., Hammond, J.P., Zelko, I., Lux, A., 2007. Zinc in plants. New
Phytol. 173, 677–702.
Bucking, H., Heyser, W., 1994. The effect of ectomycorrhizal fungi on Zn uptake and
distribution in seedlings of Pinus sylvestris L. Plant Soil 167, 203–212.
Buee, M., Vairelles, D., Garbaye, J., 2005. Year-round monitoring of diversity and
potential metabolic activity of the ectomycorrhizal community in a beech (Fagus
silvatica) forest subjected to two thinning regimes. Mycorrhiza 15, 235–245.
Campioli, M., Vincke, C., Jonard, M., Kint, V., Demaree, G., Ponette, Q., 2012. Current
status and predicted impact of climate change on forest production and biogeo-
chemistry in the temperate oceanic European zone: review and prospects for
Belgium as a case study. J. For. Res. 17, 1–18.
Cellier, M., Prive, G., Belouchi, A., Kwan, T., Rodrigues, V., Chia, W., Gros, P., 1995.
NRAMP defines a family of membrane proteins. Proc. Natl. Acad. Sci. U.S.A. 92,
10089–10093.
Chen, S., Polle, A., 2010. Salinity tolerance of Populus. Plant Biol. 12, 317–333.
Clemens, S., 2006. Toxic metal accumulation, responses to exposure and mecha-
nisms of tolerance in plants. Biochimie 88, 1707–1719.
Clemens, S., Aarts, M., Thomine, S., Verbruggen, N., 2013. Plant science: the key to
preventing slow cadmium poisoning. Trends Plant Sci. 18, 92–99.
Cocozza, C., Maiuro, L., Tognetti, R., 2011. Mapping cadmium distribution in roots
of Salicaceae through scanning electron microscopy with X-ray microanalysis.
iForest 4, 113–120.
Colpaert, J.V., Vanassche, J.A., 1992. Zinc toxicity in ectomycorrhizal Pinus sylvestris.
Plant Soil 143, 201–211.
Colpaert, J.V., Vanassche, J.A., 1993. The effects of cadmium on ectomycorrhizal Pinus
sylvestris L. New Phytol. 123, 325–333.
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, 17–24.
Courbot, M., Diez, L., Ruotolo, R., Chalot, M., Leroy, P., 2004. Cadmium-responsive
thiols in the ectomycorrhizal fungus Paxillus involutus. Appl. Environ. Microbiol.
70, 7413–7417.
Crane, S., Barkay, T., Dighton, J., 2012. The effect of mercury on the establishment of
Pinus rigida seedlings and the development of their ectomycorrhizal communi-
ties. Fungal Ecol. 5, 245–251.
Danielsen, L., Lohaus, G., Sirrenberg, A., Karlovsky, P., Bastien, C., Pilate, G., Polle, A.,
2013. Ectomycorrhizal colonization and diversity in relation to tree biomass and
nutrition in a plantation of transgenic poplars with modified lignin biosynthesis.
PLoS ONE 8, e59207.
Danielsen, L., Thurmer, A., Meinicke, P., Buee, M., Morin, E., Martin, F., Pilate, G.,
Daniel, R., Polle, A., Reich, M., 2012. Fungal soil communities in a young trans-
genic poplar plantation form a rich reservoir for fungal root communities. Ecol.
Evol. 2, 1935–1948.
Druebert, C., Lang, C., Valtanen, K., Polle, A., 2009. Beech carbon productivity as
driver of ectomycorrhizal abundance and diversity. Plant Cell Environ. 32,
992–1003.
Ducic, T., Berthold, D., Langenfeld-Heyser, R., Beese, F., Polle, A., 2009. Mycorrhizal
communities in relation to biomass production and nutrient use efficiency in
two varieties of Douglas fir (Pseudotsuga menziesii var. menziesii and var. glauca)
in different forest soils. Soil Biol. Biochem. 41, 742–753.
Ducic, T., Leinemann, L., Finkeldey, R., Polle, A., 2006. Uptake and translocation of
manganese in seedlings of two varieties of Douglas fir (Pseudotsuga menziesii
var. viridis and glauca). New Phytol. 170, 11–20.
Ducic, T., Parlade, J., Polle, A., 2008. The influence of the ectomycorrhizal fungus
Rhizopogon subareolatus on growth and nutrient element localisation in two
varieties of Douglas fir (Pseudotsuga menziesii var. menziesii and var. glauca) in
response to manganese stress. Mycorrhiza 18, 227–239.
Ducic, T., Polle, A., 2007. Manganese toxicity in two varieties of Douglas fir (Pseu-
dotsuga menziesii var. viridis and glauca) seedlings as affected by phosphorus
supply. Funct. Plant Biol. 34, 31–40.
Ducic, T., Thieme, J., Polle, A., 2012. Phosphorus compartmentalization on the cellular
level of Douglas fir root as affected by Mn toxicity: a synchrotron-based FTIR
approach. Spectr. Int. J. 27, 265–272.
Esteban, E., Deza, M.J., Zornoza, P., 2013. Kinetics of mercury uptake by oilseed rape
and white lupin: influence of Mn and Cu. Acta Physiol. Plant. 35, 2339–2344.
Fernandez, R., Bertrand, A., Casares, A., Garcia, R., Gonzalez, A., Tames, R.S., 2008.
Cadmium accumulation and its effect on the in vitro growth of woody fleabane
and mycorrhized white birch. Environ. Pollut. 152, 522–529.
Fernando, D.R., Woodrow, I.E., Jaffre, T., Dumontet, V., Marshall, A.T., Baker, A.J.M.,
2008. Foliar manganese accumulation by Maytenus founieri (Celastraceae) in
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16
14 Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
its native New Caledonian habitats: populational variation and localization by
X-ray microanalysis. New Phytol. 177, 178–185.
Ferraz, P., Fidalgo, F., Almeida, A., Teixeira, J., 2012. Phytostabilization of nickel by the
zinc and cadmium hyperaccumulator Solanum nigrum L. Are metallothioneins
involved? Plant Physiol. Biochem. 57, 254–260.
Finlay, R.D., 2008. Ecological aspects of mycorrhizal symbiosis: with special
emphasis on the functional diversity of interactions involving the extraradical
mycelium. J. Exp. Bot. 59, 1115–1126.
Flores-Monterroso, A., Canales, J., de la Torre, F., Avila, C., Canovas, F.M., 2013. Iden-
tification of genes differentially expressed in ectomycorrhizal roots during the
Pinus pinaster–Laccaria bicolor interaction. Planta 237, 1637–1650.
Fomina, M.A., Alexander, I.J., Colpaert, J.V., Gadd, G.M., 2005. Solubilization of toxic
metal minerals and metal tolerance of mycorrhizal fungi. Soil Biol. Biochem. 37,
851–866.
Gafur, A., Schutzendubel, A., Langenfeld-Heyser, R., Fritz, E., Polle, A., 2004. Compat-
ible and incompetent Paxillus involutus isolates for ectomycorrhiza formation
in vitro with poplar (Populus × canescens) differ in H2 O2 production. Plant Biol.
6, 91–99.
Gallego, S.M., Pena, L.B., Barcia, R.A., Azpilicueta, C.E., Lannone, M.F., Rosales, E.P.,
Zawoznik, M.S., Groppa, M.D., Benavides, M.P., 2012. Unravelling cadmium tox-
icity and tolerance in plants: Insight into regulatory mechanisms. Environ. Exp.
Bot. 83, 33–46.
Galli, U., Meier, M., Brunold, C., 1993. Effects of cadmium on nonmycorrhizal and
mycorrhizal Norway spruce seedlings Picea abies L. Karst and its ectomycorrhizal
fungus Laccaria laccata (Scop EX FR) BK and BR–sulfate reduction, thiols and
distribution of the heavy metal. New Phytol. 125, 837–843.
Gange, A.C., Gane, D.R.J., Chen, Y.L., Gong, M.Q., 2005. Dual colonization of Eucalyp-
tus urophylla ST Blake by arbuscular and ectomycorrhizal fungi affects levels of
insect herbivore attack. Agric. For. Entomol. 7, 253–263.
Gao, Y., Miao, C.Y., Mao, L.A., Zhou, P., Jin, Z.G., Shi, W.J., 2010. Improvement of phy-
toextraction and antioxidative defense in Solanum nigrum L. under cadmium
stress by application of cadmium-resistant strain and citric acid. J. Hazard. Mater.
181, 771–777.
Ghasemi, R., Ghaderian, S.M., Kramer, U., 2009. Interference of nickel with cop-
per and iron homeostasis contributes to metal toxicity symptoms in the nickel
hyperaccumulator plant Alyssum inflatum. New Phytol. 184, 566–580.
Godbold, D.L., Jentschke, G., Winter, S., Marschner, P., 1998. Ectomycorrhizas and
amelioration of metal stress in forest trees. Chemosphere 36, 757–762.
Gorfer, M., Persak, H., Berger, H., Brynda, S., Bandian, D., Strauss, J., 2009. Identi-
fication of heavy metal regulated genes from the root associated ascomycete
Cadophora finlandica using a genomic microarray. Mycol. Res. 113, 1377–1388.
Gustin, J.L., Loureiro, M.E., Kim, D., Na, G., Tikhonova, M., Salt, D.E., 2009. MTP1-
dependent Zn sequestration into shoot vacuoles suggests dual roles in Zn
tolerance and accumulation in Zn-hyperaccumulating plants. Plant J. 57,
1116–1127.
Hall, J.L., 2002. Cellular mechanisms for heavy metal detoxification and tolerance. J.
Exp. Bot. 53, 1–11.
Hanikenne, M., Talke, I.N., Haydon, M.J., Lanz, C., Nolte, A., Motte, P., Kroymann, J.,
Weigel, D., Kramer, U., 2008. Evolution of metal hyperaccumulation required
cis-regulatory changes and triplication of HMA4. Nature 453, 391-395.
Hartley, J., Cairney, J.W.G., Meharg, A.A., 1999. Cross-colonization of Scots pine (Pinus
sylvestris) seedlings by the ectomycorrhizal fungus Paxillus involutus in the pres-
ence of inhibitory levels of Cd and Zn. New Phytol. 142, 141–149.
Hartley-Whitaker, J., Cairney, J.W.G., Meharg, A.A., 2000. Sensitivity to Cd or Zn of
host and symbiont of ectomycorrhizal Pinus sylvestris L. (Scots pine) seedlings.
Plant Soil 218, 31–42.
Hartley, S.E., Gange, A.C., 2009. Impacts of plant symbiotic fungi on insect herbivores:
mutualism in a multitrophic context. Annu. Rev. Entomol. 54, 323–342.
He, J., Li, H., Luo, J., Ma, C.F., Li, S., Qu, L., Gai, Y., Jiang, X., Janz, D., Polle, A., Tyree,
M.T., Luo, Z.B., 2013a. A transcriptomic network underlies microstructural and
physiological responses to cadmium in Populus × canescens. Plant Physiol. 162,
424–439.
He, J.L., Ma, C.F., Ma, Y.L., Li, H., Kang, J.Q., Liu, T.X., Polle, A., Peng, C.H., Luo, Z.B.,
2013b. Cadmium tolerance in six poplar species. Environ. Sci. Pollut. Res. 20,
163–174.
He, J.L., Qin, J.J., Long, L.Y., Ma, Y.L., Li, H., Li, K., Jiang, X.N., Liu, T.X., Polle, A., Liang,
Z.S., Luo, Z.B., 2011. Net cadmium flux and accumulation reveal tissue-specific
oxidative stress and detoxification in Populus × canescens. Physiol. Plant. 143,
50–63.
Hegedus, N., Emri, T., Szilagyi, J., Karanyi, Z., Nagy, I., Penninckx, M.J., Pocsi, I., 2007.
Effect of heavy metals on the glutathione status in different ectomycorrhizal
Paxillus involutus strains. World J. Microbiol. Biotechnol. 23, 1339–1343.
Heller, G., Adomas, A., Li, G.S., Osborne, J., van Zyl, L., Sederoff, R., Finlay, R.D., Stenlid,
J., Asiegbu, F.O., 2008. Transcriptional analysis of Pinus sylvestris roots challenged
with the ectomycorrhizal fungus Laccaria bicolor. BMC Plant Biol. 8, 19.
Hewitt, E.J., 1966. Sand and water culture methods used in the study of plant nutri-
tion. Commonwealth Agricultural Bureau, Farnham Royal, UK.
Hoagland, D.R., Arnon, D.I., 1950. The Water-Culture Method for Growing Plants
Without Soil. Circular, vol. 347., 2nd ed. California Agricultural Experiment
Station, pp. 32.
Hrynkiewicz, K., Dabrowska, G., Baum, C., Niedojadlo, K., Leinweber, P., 2012. Inter-
active and single effects of ectomycorrhiza formation and Bacillus cereus on
metallothionein MT1 expression and phytoextraction of Cd and Zn by willows.
Water Air Soil Pollut. 223, 957–968.
Huang, Y., Hatayama, M., Inoue, C., 2011. Characterization of As efflux from the roots
of As hyperaccumulator Pteris vittata L. Planta 234, 1275–1284.
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
Huang, Y., Tao, S., 2004. Influences of excessive Cu on photosynthesis and growth in
ectomycorrhizal Pinus sylvestris seedlings. J. Environ. Sci. China 16, 414–419.
Huang, Y., Li, T., Huang, Z.J., Fei, Y.H., 2008. Ectomycorrhizal fungus-induced changes
of Cu and Cd speciation in the rhizosphere of Chinese pine seedlings. Pedosphere
18, 758–765.
Jamnicka, G., Bucinova, K., Havranova, I., Urban, A., 2007. Current state of mineral
nutrition and risk elements in a beech ecosystem situated near the aluminium
smelter in Ziar nad Hronom, Central Slovakia. For. Ecol. Manage. 248, 26–35.
Jean-Philippe, S.R., Franklin, J.A., Buckley, D.S., Hughes, K., 2011. The effect of mercury
on trees and their mycorrhizal fungi. Environ. Pollut. 159, 2733–2739.
Jentschke, G., Goldbold, D.L., 2000. Metal toxicity and ectomycorrhizas. Physiol.
Plant. 109, 107–116.
Jentschke, G., Marschner, P., Vodnik, D., Marth, C., Bredemeier, M., Rapp, C., Fritz, E.,
Gogala, N., Godbold, D.L., 1998. Lead uptake by Picea abies seedlings: effects of
nitrogen source and mycorrhizas. J. Plant Physiol. 153, 97–104.
Jentschke, G., Winter, S., Godbold, D.L., 1999. Ectomycorrhizas and cadmium toxicity
in Norway spruce seedlings. Tree Physiol. 19, 23–30.
Jiang, L.Y., Yang, X.E., He, Z.L., 2004. Growth response and phytoextraction of copper
at different levels in soils by Elsholtzia splendens. Chemosphere 55, 1179–1187.
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, 39–54.
Jourand, P., Ducousso, M., Reid, R., Majorel, C., Richert, C., Riss, J., Lebrun, M.,
2010. Nickel-tolerant ectomycorrhizal Pisolithus albus ultramafic ecotype iso-
lated from nickel mines in New Caledonia strongly enhance growth of the
host plant Eucalyptus globulus at toxic nickel concentrations. Tree Physiol. 30,
1311–1319.
Kaiser, B.N., Moreau, S., Castelli, J., Thomson, R., Lambert, A., Bogliolo, S., Puppo,
A., Day, D.A., 2003. The soybean NRAMP homologue, GmDMT1, is a symbi-
otic divalent metal transporter capable of ferrous iron transport. Plant J. 35,
295–304.
Kaiser, C., Koranda, M., Kitzler, B., Fuchslueger, L., Schnecker, J., Schweiger, P., Rasche,
F., Zechmeister-Boltenstern, S., Sessitsch, A., Richter, A., 2010. Belowground
carbon allocation by trees drives seasonal patterns of extracellular enzyme activ-
ities by altering microbial community composition in a beech forest soil. New
Phytol. 187, 843–858.
Kaplan, O., Ince, M., Yaman, M., 2011. Sequential extraction of cadmium in different
soil phases and plant parts from a former industrialized area. Environ. Chem.
Lett. 9, 397–404.
Kacprzak, M., Fijalkowski, K., 2009. Mycorrhiza and sewage sludge effect on biomass
of sunflower and willow during phytoremdiation of degraded terrains within
zinc foundry zone. Environ. Prot. Eng. 35, 181–187.
Khouja, H.R., Abba, S., Lacercat-Didier, L., Daghino, S., Doillon, D., Richaud, P., Martino,
E., Vallino, M., Perotto, S., Chalot, M., Blaudez, D., 2013. OmZnT1 and OmFET, two
metal transporters from the metal-tolerant strain Zn of the ericoid mycorrhizal
fungus Oidiodendron maius, confer zinc tolerance in yeast. Fungal Genet. Biol.
52, 53–64.
Kieffer, P., Dommes, J., Hoffmann, L., Hausman, J.F., Renaut, J., 2008. Quantitative
changes in protein expression of cadmium-exposed poplar plants. Proteomics
8, 2514–2530.
Kim, C.G., Power, S.A., Bell, J.N.B., 2004. Response of Pinus sylvestris seedlings to
cadmium and mycorrhizal colonisation. Water Air Soil Pollut. 155, 189–203.
Kozdroj, J., Piotrowska-Seget, Z., Krupa, P., 2007. Mycorrhizal fungi and ectomyc-
orrhiza associated bacteria isolated from an industrial desert soil protect pine
seedlings against Cd(II) impact. Ecotoxicology 16, 449–456.
Kramer, U., 2010. Metal hyperaccumulation in plants. Ann. Rev. Plant Biol. 61,
517–534.
Krpata, D., Fitz, W., Peintner, U., Langer, I., Schweiger, P., 2009. Bioconcentration
of zinc and cadmium in ectomycorrhizal fungi and associated aspen trees as
affected by level of pollution. Environ. Pollut. 157, 280–286.
Krpata, D., Peintner, U., Langer, I., Fitz, W.J., Schweiger, P., 2008. Ectomycorrhizal
communities associated with Populus tremula growing on a heavy metal con-
taminated site. Mycol. Res. 112, 1069–1079.
Krupa, P., Piotrowska-Seget, Z., 2003. Positive aspects of interaction between plants
and mycorrhizal fungi originating from soils polluted with cadmium. Polish J.
Environ. Stud. 12, 723–726.
Krupa, P., Kozdroj, J., 2004. Accumulation of heavy metals by ectomycorrhizal fungi
colonizing birch trees growing in an industrial desert soil. World J. Microbiol.
Biotechnol. 20, 427–430.
Krupa, P., Kozdroj, J., 2007. Ectomycorrhizal fungi and associated bacteria provide
protection against heavy metals in inoculated pine (Pinus sylvestris L.) seedlings.
Water Air Soil Pollut. 182, 83–90.
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, 1581–1588.
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, 2133–2141.
Kupper, H., Lombi, E., Zhao, F.J., McGrath, S.P., 2000. Cellular compartmentation
of cadmium and zinc in relation to other elements in the hyperaccumulator
Arabidopsis halleri. Planta 212, 75–84.
Lang, C., Dolynska, A., Finkeldey, R., Polle, A., 2010. Are beech (Fagus sylvatica) roots
territorial? For. Ecol. Manage. 260, 1212–1217.
Lang, C., Finkeldey, R., Polle, A., 2013. Spatial patterns of ectomycorrhizal assem-
blages in a monospecific forest in relation to host tree genotype. Front. Plant Sci.
4, http://dx.doi.org/10.3389/fpls.2013.00103.
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16
Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
Langenfeld-Heyser, R., Gao, J., Ducic, T., Tachd, P., Lu, C.F., Fritz, E., Gafur, A., Polle,
A., 2007. Paxillus involutus mycorrhiza attenuate NaCl-stress responses in the
salt-sensitive hybrid poplar Populus × canescens. Mycorrhiza 17, 121–131.
Langer, I., Krpata, D., Fitz, W.J., Wenzel, W.W., Schweiger, P.F., 2009. Zinc accumu-
lation potential and toxicity threshold determined for a metal-accumulating
Populus canescens clone in a dose-response study. Environ. Pollut. 157,
2871–2877.
Langer, I., Santner, J., Krpata, D., Fitz, W.J., Wenzel, W.W., Schweiger, P.F., 2012.
Ectomycorrhizal impact on Zn accumulation of Populus tremula L. grown in
metalliferous soil with increasing levels of Zn concentration. Plant Soil 355,
283–297.
Lanquar, V., Lelievre, F., Bolte, S., Hames, C., Alcon, C., Neumann, D., Vansuyt, G., Curie,
C., Schroder, A., Kramer, U., Barbier-Brygoo, H., Thomine, S., 2005. Mobilization
of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination
on low iron. EMBO J. 24, 4041–4051.
Lehto, T., Zwiazek, J.J., 2011. Ectomycorrhizas and water relations of trees: a review.
Mycorrhiza 21, 71–90.
Li, J., Bao, S.Q., Zhang, Y.H., Ma, X.J., Mishra-Knyrim, M., Sun, J., Sa, G., Shen, X., Polle, A.,
Chen, S.L., 2012. Paxillus involutus strains MAJ and NAU mediate K+ /Na+ homeo-
stasis in ectomycorrhizal Populus × canescens under sodium chloride stress.
Plant Physiol. 159, 1771–1786.
Lingua, G., Bona, E., Todeschini, V., Cattaneo, C., Marsano, F., Berta, G., Cavaletto, M.,
2012. Effects of heavy metals and arbuscular mycorrhiza on the leaf proteome
of a selected poplar clone: a time course analysis. PLoS ONE 7, e38662.
Lu, L.L., Tian, S.K., Zhang, J., Yang, X., Labavitch, J.M., Webb, S.M., Latimer, M.,
Brown, P.H., 2013. Efficient xylem transport and phloem remobilization of
Zn in the hyperaccumulator plant species Sedum alfredii. New Phytol. 198,
721–731.
Luo, Z.B., Janz, D., Jiang, X.N., Gobel, C., Wildhagen, H., Tan, Y.P., Rennenberg, H.,
Feussner, I., Polle, A., 2009a. Upgrading root physiology for stress tolerance
by ectomycorrhizas: Insights from metabolite and transcriptional profiling into
reprogramming for stress anticipation. Plant Physiol. 151, 1902–1917.
Luo, Z.B., Li, K., Gai, Y., Gobel, C., Wildhagen, H., Jiang, X.N., Feussner, I., Rennenberg,
H., Polle, A., 2011. The ectomycorrhizal fungus (Paxillus involutus) modulates
leaf physiology of poplar towards improved salt tolerance. Environ. Exp. Bot. 72,
304–311.
Luo, Z.B., Li, K., Jiang, X., Polle, A., 2009b. Ectomycorrhizal fungus (Paxillus involu-
tus) and hydrogels affect performance of Populus euphratica exposed to drought
stress. Ann. For. Sci. 66, 106.
Müller, A., Volmer, K., Mishra-Knyrim, M., Polle, A., 2013. Growing
poplars for research with and without mycorrhizas. Front. Plant Sci. 4,
http://dx.doi.org/10.3389/fpls.2013.00332.
Ma, Y., He, J., Ma, C.F., Luo, J., Li, H., Liu, T.X., Polle, A., Peng, C.H., Luo, Z.B., 2013.
Ectomycorrhizas with Paxillus involutus enhance cadmium uptake and tolerance
in Populus × canescens. Plant Cell Environ., http://dx.doi.org/10.1111/pce.12183.
Majorel, C., Hannibal, L., Soupe, M.E., Carriconde, F., Ducousso, M., Lebrun, M.,
Jourand, P., 2012. Tracking nickel-adaptive biomarkers in Pisolithus albus from
New Caledonia using a transcriptomic approach. Mol. Ecol. 21, 2208–2223.
Marschner, P., Godbold, D.L., Jentschke, G., 1996. Dynamics of lead accumulation in
mycorrhizal and non-mycorrhizal Norway spruce (Picea abies (L) Karst). Plant
Soil 178, 239–245.
Martin, F., et al., 2008. The genome of Laccaria bicolor provides insights into mycor-
rhizal symbiosis. Nature 452, 88-92.
Martin, F., Kohler, A., Duplessis, S., 2007. Living in harmony in the wood under-
ground: ectomycorrhizal genomics. Curr. Opin. Plant Biol. 10, 204–210.
Martin, F., et al., 2010. Perigord black truffle genome uncovers evolutionary origins
and mechanisms of symbiosis. Nature 464, 1033–1038.
Martin, F., Nehls, U., 2009. Harnessing ectomycorrhizal genomics for ecological
insights. Curr. Opin. Plant Biol. 12, 508–515.
Mayor, D.J., Gray, N.B., Elver-Evans, J., Midwood, A.J., Thornton, B., 2013.
Metal-macrofauna interactions determine microbial community structure and
function in copper contaminated sediments. PLoS ONE 8, e64940.
Meharg, A.A., 2003. The mechanistic basis of interactions between mycorrhizal asso-
ciations and toxic metal cations. Mycol. Res. 107, 1253–1265.
Mendoza-Cozatl, D.G., Jobe, T.O., Hauser, F., Schroeder, J.I., 2011. Long-distance
transport, vacuolar sequestration, tolerance, and transcriptional responses
induced by cadmium and arsenic. Curr. Opin. Plant Biol. 14, 554–562.
Merkle, S.A., 2006. Engineering forest trees with heavy metal resistance genes. Silvae
Genetica 55, 263–268.
Migeon, A., Blaudez, D., Wilkins, O., Montanini, B., Campbell, M.M., Richaud, P.,
Thomine, S., Chalot, M., 2010. Genome-wide analysis of plant metal transporters,
with an emphasis on poplar. Cell. Mol. Life Sci. 67, 3763–3784.
Milner, M.J., Craft, E., Yamaji, N., Koyama, E., Ma, J.F., Kochian, L.V., 2012. Character-
ization of the high affinity Zn transporter from Noccaea caerulescens, NcZNT1,
and dissection of its promoter for its role in Zn uptake and hyperaccumulation.
New Phytol. 195, 113–123.
Milner, M.J., Kochian, L.V., 2008. Investigating heavy-metal hyperaccumulation
using Thlaspi caerulescens as a model system. Ann. Bot. 102, 3–13.
Mok, T., Chen, J.S., Shlykov, M.A., Saier, M.H., 2012. Bioinformatic analyses of bacterial
mercury ion (Hg2+ ) Transporters. Water Air Soil Pollut. 223, 4443–4457.
Mrnka, L., Kuchar, M., Cieslarova, Z., Matejka, P., Szakova, J., Tlustos, P., Vosatka, 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, 399–410.
Muller, L.A.H., Craciun, A.R., Ruytinx, J., Lambaerts, M., Verbruggen, N., Vangronsveld,
J., Colpaert, J.V., 2007a. Gene expression profiling of a Zn-tolerant and a
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
15
Zn-sensitive Suillus luteus isolate exposed to increased external zinc concen-
trations. Mycorrhiza 17, 571–580.
Muller, T., Avolio, M., Olivi, M., Benjdia, M., Rikirsch, E., Kasaras, A., Fitz, M., Chalot,
M., Wipf, D., 2007b. Nitrogen transport in the ectomycorrhiza association: the
Hebeloma cylindrosporum–Pinus pinaster model. Phytochemistry 68, 41–51.
Nehls, U., Gohringer, F., Wittulsky, S., Dietz, S., 2010. Fungal carbohydrate support
in the ectomycorrhizal symbiosis: a review. Plant Biol. 12, 292–301.
Nerg, A.-M., Kasurinen, A., Holopainen, T., Julkunen-Tiitto, R., Neuvonen, S.,
Holopainen, J.K., 2009. Interactions of ectomycorrhizas and above-ground insect
herbivores on silver birch. Plant Signal. Behav. 4, 355–357.
Nerg, A.M., Kasurinen, A., Holopainen, T., Julkunen-Tiitto, R., Neuvonen, S.,
Holopainen, J.K., 2008. The significance of ectomycorrhizas in chemical quality
of silver birch foliage and above-ground insect herbivore performance. J. Chem.
Ecol. 34, 1322–1330.
Niemeyer, J.C., Nogueira, M.A., Carvalho, G.M., Cohin-De-Pinho, S.J., Outeiro, U.S.,
Rodrigues, G.G., da Silva, E.M., Sousa, J.P., 2012. Functional and structural param-
eters to assess the ecological status of a metal contaminated area in the tropics.
Ecotox. Environ. Saf. 86, 188–197.
Nystedt, B., et al., 2013. The Norway spruce genome sequence and conifer genome
evolution. Nature 497, 579–584.
Osakabe, Y., Kajita, S., Osakabe, K., 2011. Genetic engineering of woody plants:
current and future targets in a stressful environment. Physiol. Plant. 142,
105–117.
Ott, T., Fritz, E., Polle, A., Schutzendubel, A., 2002. Characterisation of antioxida-
tive systems in the ectomycorrhiza-building basidiomycete Paxillus involutus
(Bartsch) Fr. and its reaction to cadmium. FEMS Microbiol. Ecol. 42,
359–366.
Papoyan, A., Kochian, L.V., 2004. Identification of Thlaspi caerulescens genes that
may be involved in heavy metal hyperaccumulation and tolerance. Charac-
terization of a novel heavy metal transporting ATPase. Plant Physiol. 136,
3814–3823.
Park, J., Song, W.Y., Ko, D., Eom, Y., Hansen, T.H., Schiller, M., Lee, T.G., Martinoia,
E., Lee, Y., 2012. The phytochelatin transporters AtABCC1 and AtABCC2 mediate
tolerance to cadmium and mercury. Plant J. 69, 278–288.
Patra, M., Bhowmik, N., Bandopadhyay, B., Sharma, A., 2004. Comparison of
mercury, lead and arsenic with respect to genotoxic effects on plant sys-
tems and the development of genetic tolerance. Environ. Exp. Bot. 52,
199–223.
Pauwels, M., Willems, G., Roosens, N., Frerot, H., Saumitou-Laprade, P., 2008. Merging
methods in molecular and ecological genetics to study the adaptation of plants
to anthropogenic metal-polluted sites: implications for phytoremediation. Mol.
Ecol. 17, 108–119.
Peiter, E., Montanini, B., Gobert, A., Pedas, P., Husted, S., Maathuis, F.J.M., Blaudez,
D., Chalot, M., Sanders, D., 2007. A secretory pathway-localized cation diffusion
facilitator confers plant manganese tolerance. Proc. Natl. Acad. Sci. U.S.A. 104,
8532–8537.
Pena, R., Offermann, C., Simon, J., Naumann, P.S., Gessler, A., Holst, J., Dannenmann,
M., Mayer, H., Kogel-Knabner, I., Rennenberg, H., Polle, A., 2010. Girdling affects
ectomycorrhizal fungal (EMF) diversity and reveals functional differences in
EMF community composition in a beech forest. Appl. Environ. Microbiol. 76,
1831–1841.
Pena, R., Polle, A., 2013. Attributing functions to ectomycorrhizal fungal
identities in assemblages for nitrogen acquisition under stress. ISME J.,
http://dx.doi.org/10.1038/ismej.2013.158.
Pena, R., Simon, J., Rennenberg, H., Polle, A., 2013. Ectomycorrhiza affect architecture
and nitrogen partitioning of beech (Fagus sylvatica L.) seedlings under shade and
drought. Environ. Exp. Bot. 87, 207–217.
Pilon, M., Cohu, C.M., Ravet, K., Abdel-Ghany, S.E., Gaymard, F., 2009. Essential tran-
sition metal homeostasis in plants. Curr. Opin. Plant Biol. 12, 347–357.
Plassard, C., Dell, B., 2010. Phosphorus nutrition of mycorrhizal trees. Tree Physiol.
30, 1129–1139.
Plett, J.M., Martin, F., 2011. Blurred boundaries: lifestyle lessons from ectomycor-
rhizal fungal genomes. Trends Genet. 27, 14–22.
Polle, A., Klein, T., Kettner, C., 2013. Impact of cadmium on young plants of Populus
euphratica and P. × canescens, two poplar species that differ in stress tolerance.
New For. 44, 13–22.
Pritsch, K., Esperschuetz, J., Haesler, F., Raidl, S., Winkler, B., Schloter, M., 2009.
Structure and activities of ectomycorrhizal and microbial communities in the
rhizosphere of Fagus sylvatica under ozone and pathogen stress in a lysimeter
study. Plant Soil 323, 97–109.
Rajkumar, M., Sandhya, S., Prasad, M.N.V., Freitas, H., 2012. Perspectives of plant-
associated microbes in heavy metal phytoremediation. Biotechnol. Adv. 30,
1562–1574.
Ray, P., Adholeya, A., 2009. Correlation between organic acid exudation and metal
uptake by ectomycorrhizal fungi grown on pond ash in vitro. Biometals 22,
275–281.
Rooney, D.C., Killham, K., Bending, G.D., Baggs, E., Weih, M., Hodge, A., 2009.
Mycorrhizas and biomass crops: opportunities for future sustainable develop-
ment. Trends Plant Sci. 14, 542–549.
Rotkittikhun, P., Kruatrachue, M., Chaiyarat, R., Ngernsansaruay, C., Pokethitiyook,
P., Paijitprapaporn, A., Baker, A.J.M., 2006. Uptake and accumulation of lead
by plants from the Bo Ngam lead mine area in Thailand. Environ. Pollut. 144,
681–688.
Schützendübel, A., Polle, A., 2002. Plant responses to abiotic stresses: heavy metal-
induced oxidative stress and protection by mycorrhization. J. Exp. Bot. 53,
1351–1365.
 ARTICLE IN PRESS
G Model
EEB-2698; No. of Pages 16
16 Z.-B. Luo et al. / Environmental and Experimental Botany xxx (2013) xxx–xxx
Sell, J., Kayser, A., Schulin, R., Brunner, I., 2005. Contribution of ectomycorrhizal fungi
to cadmium uptake of poplars and willows from a heavily polluted soil. Plant
Soil 277, 245–253.
Seth, C.S., Remans, T., Keunen, E., Jozefczak, M., Gielen, H., Opdenakker, K., Weyens,
N., Vangronsveld, J., Cuypers, A., 2012. Phytoextraction of toxic metals: a central
role for glutathione. Plant Cell Environ. 35, 334–346.
Sharma, S.S., Dietz, K.J., 2009. The relationship between metal toxicity and cellular
redox imbalance. Trends Plant Sci. 14, 43–50.
Shim, D., Kim, S., Choi, Y.I., Song, W.Y., Park, J., Youk, E.S., Jeong, S.C., Martinoia,
E., Noh, E.W., Lee, Y., 2013. Transgenic poplar trees expressing yeast cadmium
factor 1 exhibit the characteristics necessary for the phytoremediation of mine
tailing soil. Chemosphere 90, 1478–1486.
Shiyab, S., Chen, J., Han, F.X.X., Monts, D.L., Matta, F.B., Gu, M.M., Su, Y., Masad, M.A.,
2009. Mercury-induced oxidative stress in indian mustard (Brassica juncea L.).
Environ. Toxicol. 24, 462–471.
Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis, 3rd ed. Academic Press, London.
Solti, A., Sarvari, E., Toth, B., Basa, B., Levai, L., Fodor, F., 2011. Cd affects the translo-
cation of some metals either Fe-like or Ca-like way in poplar. Plant Physiol.
Biochem. 49, 494–498.
Sousa, N.R., Ramos, M.A., Marques, A., Castro, P.M.L., 2012. The effect of ectomy-
corrhizal fungi forming symbiosis with Pinus pinaster seedlings exposed to
cadmium. Sci. Total Environ. 414, 63–67.
Takahashi, R., Ishimaru, Y., Senoura, T., Shimo, H., Ishikawa, S., Arao, T., Nakanishi,
H., Nishizawa, N.K., 2011. The OsNRAMP1 iron transporter is involved in Cd
accumulation in rice. J. Exp. Bot. 62, 4843–4850.
Takahashi, R., Ishimaru, Y., Shimo, H., Ogo, Y., Senoura, T., Nishizawa, N.K., Nakanishi,
H., 2012. The OsHMA2 transporter is involved in root-to-shoot translocation of
Zn and Cd in rice. Plant Cell Environ. 35, 1948–1957.
Talke, I.N., Hanikenne, M., Kramer, U., 2006. Zinc-dependent global transcriptional
control, transcriptional deregulation, and higher gene copy number for genes
in metal homeostasis of the hyperaccumulator Arabidopsis halleri. Plant Physiol.
142, 148–167.
Thomine, S., Lelievre, F., Debarbieux, E., Schroeder, J.I., Barbier-Brygoo, H., 2003.
AtNRAMP3, a multispecific vacuolar metal transporter involved in plant
responses to iron deficiency. Plant J. 34, 685–695.
Turnau, K., Kottke, I., Dexheimer, J., 1996. Toxic element filtering in Rhizopogon rose-
olus Pinus sylvestris mycorrhizas collected from calamine dumps. Mycol. Res.
100, 16–22.
Turnau, K., Mleczko, P., Blaudez, D., Chalot, M., Botton, B., 2002. Heavy metal binding
properties of Pinus sylvestris mycorrhizas from industrial wastes. Acta Soc. Bot.
Pol. 71, 253–261.
Turnau, K., Przybylowicz, W.J., Mesjasz-Przybyowicz, J., 2001. Heavy metal distri-
bution in Suillus luteus mycorrhizas – as revealed by micro-PIXE analysis. Nucl.
Instrum. Methods Phys. Res. Sect. B: Beam Interact. Mater. Atoms 181, 649–658.
Tuskan, G.A., et al., 2006. The genome of black cottonwood, Populus trichocarpa (Torr.
& Gray). Science 313, 1596–1604.
Ueno, D., Milner, M.J., Yamaji, N., Yokosho, K., Koyama, E., Zambrano, M.C., Kaskie,
M., Ebbs, S., Kochian, L.V., Ma, J.F., 2011. Elevated expression of TcHMA3 plays
a key role in the extreme Cd tolerance in a Cd-hyperaccumulating ecotype of
Thlaspi caerulescens. Plant J. 66, 852–862.
Utmazian, M., Schweiger, P., Sommer, P., Gorfer, M., Strauss, J., Wenzel, W.W., 2007.
Influence of Cadophora finlandica and other microbial treatments on cadmium
and zinc uptake in willows grown on polluted soil. Plant Soil Environ. 53,
158–166.
Please cite this article in press as: Luo, Z.-B., et al., The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot.
(2013), http://dx.doi.org/10.1016/j.envexpbot.2013.10.018
van der Ent, A., Baker, A.J.M., Reeves, R.D., Pollard, A.J., Schat, H., 2013. Hyperaccu-
mulators of metal and metalloid trace elements: Facts and fiction. Plant Soil 362,
319–334.
Van Tichelen, K.K., Colpaert, J.V., Vangronsveld, J., 2001. Ectomycorrhizal protection
of Pinus sylvestris against copper toxicity. New Phytol. 150, 203–213.
Vazquez, M.D., Barcelo, J., Poschenrieder, C., Madico, J., Hatton, P., Baker, A.J.M., Cope,
G.H., 1992. Localization of zinc and cadmium in Thlaspi-Caerulescens (Brassi-
caceae), a metallophyte that can hyperaccumulate both metals. J. Plant Physiol.
140, 350–355.
Venkatachalam, P., Srivastava, A.K., Raghothama, K.G., Sahi, S.V., 2009. Genes
induced in response to mercury-ion-exposure in heavy metal hyperaccumulator
Sesbania drummondii. Environ. Sci. Technol. 43, 843–850.
Visoottiviseth, P., Francesconi, K., Sridokchan, W., 2002. The potential of Thai indige-
nous plant species for the phytoremediation of arsenic contaminated land.
Environ. Pollut. 118, 453–461.
Vollenweider, P., Cosio, C., Gunthardt-Goerg, M.S., Keller, C., 2006. Localization and
effects of cadmium in leaves of a cadmium-tolerant willow (Salix viminalis L.).
Part II. Microlocalization and cellular effects of cadmium. Environ. Exp. Bot. 58,
25–40.
Vollenweider, P., Menard, T., Gunthardt-Goerg, M.S., 2011. Compartmentation of
metals in foliage of Populus tremula grown on soils with mixed contamination.
I. From the tree crown to leaf cell level. Environ. Pollut. 159, 324–336.
Walker, R.F., McLaughlin, S.B., West, D.C., 2004. Establishment of sweet birch on
surface mine spoil as influenced by mycorrhizal inoculation and fertility. Restor.
Ecol. 12, 8–19.
Wang, H., Shan, X.Q., Wen, B., Zhang, S.Z., Wang, Z.J., 2004. Responses of antiox-
idative enzymes to accumulation of copper in a copper hyperaccumulator of
Commoelina communis. Arch. Environ. Contam. Toxicol. 47, 185–192.
Werner, A., Karolewski, P., 2004. The effects of toxic metals, content of nutrients and
inoculation with mycorrhizal fungi on the level of phenolics in roots and growth
of Scots pine seedlings. Act. Physiol. Plant. 26, 177–186.
Werner, A., Napierala-Filipiak, A., Mardarowicz, M., Gawdzik, J., 2004. The effects of
heavy metals, content of nutrients and inoculation with mycorrhizal fungi on
the level of terpenoids in roots of Pinus sylvestris seedlings. Act. Physiol. Plant.
26, 187–196.
Xiao, H.H., Yin, L.P., Xu, X.F., Li, T.Z., Han, Z.H., 2008. The iron-regulated transporter,
MbNRAMP1, isolated from Malus baccata is involved in Fe, Mn and Cd trafficking.
Ann. Bot. 102, 881–889.
Xue, S.G., Chen, Y.X., Reeves, R.D., Baker, A.J.M., Lin, Q., Fernando, D.R., 2004. Man-
ganese uptake and accumulation by the hyperaccumulator plant Phytolacca
acinosa Roxb. (Phytolaccaceae). Environ. Pollut. 131, 393–399.
Yamaguchi, A., Tamang, D.G., Saier, M.H., 2007. Mercury transport in bacteria. Water
Air Soil Pollut. 182, 219–234.
Zacchini, M., Pietrini, F., Mugnozza, G.S., Iori, V., Pietrosanti, L., Massacci, A., 2009.
Metal tolerance, accumulation and translocation in poplar and willow clones
treated with cadmium in hydroponics. Water Air Soil Pollut. 197, 23–34.
Zhao, F.J., Jiang, R.F., Dunham, S.J., McGrath, S.P., 2006. Cadmium uptake, transloca-
tion and tolerance in the hyperaccumulator Arabidopsis halleri. New Phytol. 172,
646–654.
Zhao, F.J., McGrath, S.P., 2009. Biofortification and phytoremediation. Curr. Opin.
Plant Biol. 12, 373–380.
Zheng, W.S., Fei, Y.H., Huang, Y., 2009. Soluble protein and acid phosphatase exuded
by ectomycorrhizal fungi and seedlings in response to excessive Cu and Cd. J.
Environ. Sci. 21, 1667–1672.