Download Heavy metal hyperaccumulating plants: How and why do they do it

Plant Science 180 (2011) 169–181
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Plant Science
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Review
Heavy metal hyperaccumulating plants: How and why do they do it?
And what makes them so interesting?
Nicoletta Rascio a,∗ , Flavia Navari-Izzo b
a
b
Department of Biology, University of Padova, via U. Bassi 58/B, I-35121 Padova, Italy
Department of Chemistry and Agricultural Biotechnologies, University of Pisa, via San Michele degli Scalzi 2, I-56124 Pisa, Italy
a r t i c l e
i n f o
Article history:
Received 26 May 2010
Received in revised form 25 August 2010
Accepted 26 August 2010
Available online 15 September 2010
Keywords:
Heavy metal uptake
Heavy metal translocation
Heavy metal detoxification/sequestration
Hyperaccumulators
Phytomining
Phytoremediation
a b s t r a c t
The term “hyperaccumulator” describes a number of plants that belong to distantly related families,
but share the ability to grow on metalliferous soils and to accumulate extraordinarily high amounts of
heavy metals in the aerial organs, far in excess of the levels found in the majority of species, without suffering phytotoxic effects. Three basic hallmarks distinguish hyperaccumulators from related
non-hyperaccumulating taxa: a strongly enhanced rate of heavy metal uptake, a faster root-to-shoot
translocation and a greater ability to detoxify and sequester heavy metals in leaves. An interesting
breakthrough that has emerged from comparative physiological and molecular analyses of hyperaccumulators and related non-hyperaccumulators is that most key steps of hyperaccumulation rely on
different regulation and expression of genes found in both kinds of plants. In particular, a determinant
role in driving the uptake, translocation to leaves and, finally, sequestration in vacuoles or cell walls of
great amounts of heavy metals, is played in hyperaccumulators by constitutive overexpression of genes
encoding transmembrane transporters, such as members of ZIP, HMA, MATE, YSL and MTP families.
Among the hypotheses proposed to explain the function of hyperaccumulation, most evidence has supported the “elemental defence” hypothesis, which states that plants hyperaccumulate heavy metals as a
defence mechanism against natural enemies, such as herbivores. According to the more recent hypothesis of “joint effects”, heavy metals can operate in concert with organic defensive compounds leading to
enhanced plant defence overall.
Heavy metal contaminated soils pose an increasing problem to human and animal health. Using plants
that hyperaccumulate specific metals in cleanup efforts appeared over the last 20 years. Metal accumulating species can be used for phytoremediation (removal of contaminant from soils) or phytomining
(growing plants to harvest the metals). In addition, as many of the metals that can be hyperaccumulated
are also essential nutrients, food fortification and phytoremediation might be considered two sides of the
same coin. An overview of literature discussing the phytoremediation capacity of hyperaccumulators to
clean up soils contaminated with heavy metals and the possibility of using these plants in phytomining
is presented.
© 2010 Elsevier Ireland Ltd. All rights reserved.
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
What are heavy metal hyperaccumulator plants? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
How do plants hyperaccumulate heavy metals? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Heavy metal uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Root-to-shoot translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Detoxification/sequestration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Why did plants evolve hyperaccumulation of heavy metals? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
The “elemental defence” hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
The “joint effects” hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author. Tel.: +39 049 8276278; fax: +39 049 8276260.
E-mail address: [email protected] (N. Rascio).
0168-9452/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2010.08.016
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5.
6.
N. Rascio, F. Navari-Izzo / Plant Science 180 (2011) 169–181
Why do hyperaccumulators attract so much interest? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Potential application in phytoremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Transfer of hyperaccumulation traits to rapidly growing species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Potential application in phytomining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
From a chemical point of view, the term heavy metal is strictly
ascribed to transition metals with atomic mass over 20 and specific
gravity above 5. In biology, “heavy” refers to a series of metals and
also metalloids that can be toxic to both plants and animals even at
very low concentrations. Here the term “heavy metals” will be for
these potentially phytotoxic elements.
Some of these heavy metals, such as As, Cd, Hg, Pb or Se, are
not essential, since they do not perform any known physiological
function in plants. Others, such as Co, Cu, Fe, Mn, Mo, Ni and Zn,
are essential elements required for normal growth and metabolism
of plants. These latter elements can easily lead to poisoning when
their concentration rises to supra-optimal values. Heavy metal
phytotoxicity may result from alterations of numerous physiological processes caused at cellular/molecular level by inactivating
enzymes, blocking functional groups of metabolically important
molecules, displacing or substituting for essential elements and
disrupting membrane integrity. A rather common consequence
of heavy metal poisoning is the enhanced production of reactive
oxygen species (ROS) due to interference with electron transport activities, especially that of chloroplast membranes [1,2]. This
increase in ROS exposes cells to oxidative stress leading to lipid
peroxidation, biological macromolecule deterioration, membrane
dismantling, ion leakage, and DNA-strand cleavage [3–5]. Plants
resort to a series of defence mechanisms that control uptake,
accumulation and translocation of these dangerous elements and
detoxify them by excluding the free ionic forms from the cytoplasm
(Fig. 1). One commonly employed strategy lies in hindering the
entrance of heavy metals into root cells through entrapment in the
apoplastic environment by binding them to exuded organic acids
[6] or to anionic groups of cell walls [7,8]. Most of the heavy metals that do enter the plant are then kept in root cells, where they
are detoxified by complexation with amino acids, organic acids or
metal-binding peptides and/or sequestered into vacuoles [9]. This
greatly restricts translocation to the above-ground organs thus protecting the leaf tissues, and particularly the metabolically active
photosynthetic cells from heavy metal damage. A further defence
mechanism generally adopted by heavy metal-exposed plants is
enhancement of cell antioxidant systems which counteracts oxidative stress [4,10].
It is interesting to notice that there are plants that survive, grow
and reproduce on natural metalliferous soils as well as on sites polluted with heavy metals as a result of anthropogenic activities. The
majority of species that tolerate heavy metal concentrations that
are highly toxic to the other plants behave as “excluders” (Fig. 1),
relying on tolerance and even hypertolerance strategies helpful for
restricting metal entrance. They retain and detoxify most of the
heavy metals in the root tissues, with a minimized translocation to
the leaves whose cells remain sensitive to the phytotoxic effects [9].
Nevertheless, a number of hypertolerant species, defined as “hyperaccumulators”, exhibit an opposite behaviour as far as heavy metal
uptake and distribution in the plant is concerned (Fig. 1).
2. What are heavy metal hyperaccumulator plants?
The term “hyperaccumulator” was coined [11] for plants (Fig. 1)
that, differently from the excluder plants, actively take up exceed-
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ingly large amounts of one or more heavy metals from the
soil. Moreover, the heavy metals are not retained in the roots
but are translocated to the shoot and accumulated in aboveground organs, especially leaves, at concentrations 100–1000-fold
higher than those found in non-hyperaccumulating species. They
show no symptoms of phytotoxicity [12,13]. Although a distinct feature, hyperaccumulation also relies on hypertolerance,
an essential key property allowing plants to avoid heavy metal
poisoning, to which hyperaccumulator plants are as sensitive as
non-hyperaccumulators [14].
About 450 angiosperm species have been identified so far as
heavy metal (As, Cd, Co, Cu, Mn, Ni, Pb, Sb, Se, Tl, Zn,) hyperaccumulators, accounting for less than 0.2% of all known species.
However, new reports of this kind of plants continue to accrue
[15–18], so that it is conceivable that many yet unidentified hyperaccumulators may occur in nature. On the other hand, some species
classified as hyperaccumulators on the basis of field samples might
be deleted from the list if this trait is unconfirmed by experimentation under controlled conditions [19]. For instance, the finding that
in a number of cuprophytes the Cu and Co hyperaccumulation by
field samples was actually due to leaf surface contamination has
Fig. 1. Mechanisms involved in heavy metal hypertolerance and heavy metal distribution in an excluder non-hyperaccumulator (left) and a hyperaccumulator (right)
plant. (1) Heavy metal binding to the cell walls and/or cell exudates, (2) root uptake,
(3) chelation in the cytosol and/or sequestration in vacuoles, (4) root-to-shoot
translocation. The spots indicate the plant organ in which the different mechanisms occur and the spot sizes the level of each of them. According to the elemental
defence hypothesis the high heavy metal concentrations make hyperaccumulator
leaves poisonous to herbivores.
N. Rascio, F. Navari-Izzo / Plant Science 180 (2011) 169–181
led to a critical re-examination of the Cu/Co hyperaccumulators
[20,21].
The hyperaccumulator species are distributed in a wide range
of distantly related families, showing that the hyperaccumulation
trait has evolved independently more than once under the spur of
selective ecological factors. The evolutionary reasons that gave rise
to hyperaccumulating plants are unknown and still under debate.
However, a series of hypotheses have been proposed. They will be
discussed in the following section. Heavy metal hyperacumulators
do occur on metal-rich soils in both tropical and temperate zones.
They are found in vegetations from regions of South Africa, New
Caledonia, Latin America as well as of North America and Europe
[22].
Initially the term hyperaccumulator referred to plants able to
accumulate more than 1 mg g−1 Ni (dry weight) in the shoot, an
exceptionally high heavy metal concentration considering that in
vegetative organs of most plants Ni toxicity starts from 10 to
15 ␮g g−1 . Threshold values were successively provided to define
the hyperaccumulation of each other heavy metal, based on its
specific phytotoxicity. According to such a criterion hyperaccumulators are plants that, when growing on native soils, concentrate
>10 mg g−1 (1%) Mn or Zn, >1 mg g−1 (0.1%) As, Co, Cr, Cu, Ni, Pb, Sb,
Se or Tl, and >0.1 mg g−1 (0.01%) Cd in the aerial organs, without
suffering phytotoxic damage [23]. Ni is hyperaccumulated by the
greatest number of taxa (more than 75%), while a low number of
hyperaccumulators (only 5 species to date) has been found for Cd,
which is one of the most toxic heavy metals. Ni is also the metal
that has been shown to reach the highest concentration in a plant.
This occurs in Sebertia acuminata (Sapotaceae), a tree endemic to
the serpentine soil from New Caledonia, which accumulates up to
26% Ni (dry mass) in its latex [24]. About 25% of discovered hyperaccumulators belong to the family of Brassicaceae and, in particular,
to genera Thlaspi and Alyssum. These also include the highest number of Ni hyperaccumulating taxa [25]. Zn hyperaccumulators are
less numerous and include Arabidopsis halleri and species of Thlaspi,
among Brassicaceae [22], and Sedum alfredii (Crassulaceae) [26].
A. halleri and S. alfredii, together with Thlaspi caerulescens and T.
praecox, are the four recognized species that, besides Zn, hyperaccumulate Cd. Recently Solanum nigrum (Solanaceae) has been
noticed as the fifth Cd hyperaccumulator [16]. Species hyperaccumulating Se are distributed in genera of different families, among
which Fabaceae, Asteraceae, Rubiaceae, Brassicaceae, Scrophulariaceae and Chenopodiaceae [27]. Besides some angiosperms, such
as the Brassicaceae Isatis cappadocica and Hesperis persica [18,28], a
number of brake ferns belonging to the genus Pteris have also been
found to hyperaccumulate As [29,30].
Most hyperaccumulators are endemic to metalliferous soils
behaving as “strict metallophytes”, whereas some “facultative
metallophytes” can live also on non-metalliferous ones, although
are more prevalent on metal-enriched habitats [31]. Furthermore, there are species that include both metallicolous and
non-metallicolous populations. In some of these, such as in Zn
hyperaccumulators A. halleri and T. caerulescens, the hyperaccumulation is a constitutive trait at the species level, being found in all
populations [32,33]. In others, such as in the Zn hyperaccumulator S. alfredii and in Cd hyperaccumulators, this trait, instead, is not
constitutive at the species level, but only confined to metallicolous
populations [23,34,35].
3. How do plants hyperaccumulate heavy metals?
The degree of hyperaccumulation of one or more heavy metals
can vary significantly in different species or also in populations and
ecotypes of the same species [36,37]. However, hyperaccumulation
depends on three basic hallmarks that distinguish hyperaccumula-
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tors from related non-hyperaccumulator taxa. These common traits
are: a much greater capability of taking up heavy metals from the
soil; a faster and effective root-to-shoot translocation of metals;
and a much greater ability to detoxify and sequester huge amounts
of heavy metals in the leaves (Fig. 1). Significant progress in understanding the mechanisms governing metal hyperaccumulation has
been made in the last decade through comparative physiological,
genomic, and proteomic studies of hyperaccumulators and related
non-hyperaccumulator plants. A great number of studies are on
T. caerulescens and A. halleri, which have become model plants
[38–40]. A very interesting feature revealed by this research is that
most key steps in hyperaccumulation do not rely on novel genes,
but depend on genes common to hyperaccumulators and nonhyperaccumulators, that are differently expressed and regulated
in the two kinds of plants [23].
3.1. Heavy metal uptake
Comparative studies have revealed that the enhanced Zn uptake
into T. caerulescens and A. halleri roots, in comparison to congener non-hyperaccumulator species, can be attributed to the
constitutive overexpression of some genes belonging to the ZIP
(Zinc-regulated transporter Iron-regulated transporter Proteins)
family, coding for plasma membrane located cation transporters
[41] (Fig. 2). Moreover, the expression of these ZIP genes (ZTN1
and ZTN2 in T. caerulescens and ZIP6 and ZIP9 in A. halleri), that in
non-hyperaccumulating plants is Zn-regulated [42] and occurs at
detectable levels only under Zn deficiency, in hyperaccumulators
is irrespective of Zn supply still persisting at high Zn availability
[41,43].
The decreasing uptake of Cd by roots supplied with increasing
Zn concentration, found in Cd/Zn hyperaccumulator A. halleri and in
most ecotypes of T. caerulescens, clearly demonstrates that Cd influx
is largely due to Zn transporters (Fig. 2), with a strong preference for
Zn over Cd [44]. Surprisingly, in plants of the Ganges ecotype of T.
caerulescens, which exhibit an exceptionally high ability to hyperaccumulate Cd in aerial tissues, Cd uptake is not inhibited by Zn,
thus suggesting the presence in root cells of a specific and efficient
independent Cd transport system [45]. The supposed existence of a
transporter specific to this metal, regarded as unessential, raises the
question as to whether Cd might play some physiological roles in
that T. caerulescens accession. In shoots of the Ganges plants a positive correlation between Cd concentration and carbonic anhydrase
activity has been found [46]. The only physiological function of this
heavy metal had previously been noticed in the marine diatom Thalassiosira weissglogii owing to its finding in the active metal-binding
site of a peculiar Cd-containing carbonic anhydrase [47,48].
Specific transporters for Ni hyperaccumulation have not yet
been recognized. However, the preference of Zn over Ni by some
Zn/Ni hyperaccumulators supplied with the same concentration of
both heavy metals strongly suggests that a Zn transport system
(Fig. 2) might also be employed in Ni entrance into roots [49].
Considerable evidence exists that As can enter plant roots as
arsenate via transporters of the chemical analogue phosphate [50]
(Fig. 2). In root cells of As hyperaccumulator Pteris vittata plasma
membranes have a higher density of phosphate/arsenate transporters than non-hyperaccumulator P. tremula, plausibly due to
constitutive gene overexpression [51]. Furthermore, the enhanced
As uptake by the hyperaccumulating fern depends on the higher
affinity for arsenate by the phosphate/arsenate transport systems
[52] as well as on the plant’s ability to increase As bioavailability in
the rhizosphere by reducing pH via root exudation of large amounts
of dissolved organic carbon [53]. The pH decrease, in fact, enhances
the water soluble As that can be taken up by the roots [53,54].
The chemical similarity between sulphate and selenate accounts
for the root uptake of Se in this form through high-affinity sulphate
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Fig. 2. A scheme showing transport systems constitutively overexpressed and/or with enhanced affinity to heavy metals, which are though to be involved in uptake, root-toshoot translocation and heavy metal sequestration traits of hyperaccumulator plants. (CAX = Cation Exchangers; CDF = Cation Diffusion Facilitators; FDR3 = a member of the
Multidrug and Toxin Efflux family; HM = Heavy Metals; HMA = Heavy Metal transporting ATPases; NA = Nicotinamine; NIP = Nodulin 26-like Intrinsic Proteins; P = Phosphate
transporters; S = Sulphate transporters; YSL = Yellow Strip 1-Like Proteins; ZIP = Zinc-regulated transporter Iron-regulated transporter Proteins). For details and references
see the text.
transporters (Fig. 2), whose activity is regulated by the S status of
the plant [55,56]. In Se hyperaccumulators, such as Astragalus bisulcatus (Fabaceae) and Stanleya pinnata (Brassicaceae), the Se/S ratios
in shoots are much higher than in non-hyperaccumulator sister
species. This supports the idea of a role in this increased Se uptake
of one or more sulphate transporters, which may have acquired a
Se-specificity, becoming independent of the plant S status [57].
3.2. Root-to-shoot translocation
Differently from non-hyperaccumulator plants, which retain
in root cells most of the heavy metal taken up from the soil,
detoxifying them by chelation in the cytoplasm or storing them
into vacuoles, hyperaccumulators rapidly and efficiently translocate these elements to the shoot via the xylem (Fig. 1). This
entails, of course, the heavy metal availability for xylem loading, which derives from a low sequestration into and a ready
efflux out of the vacuoles, plausibly due to specific features of
root cell tonoplast [58]. As a matter of fact the amount of Zn
sequestered into cell root vacuoles is 2–3-fold lower and the
Zn efflux out of vacuoles almost twice as fast in the hyperaccumulators T. caerulescens [58] and S. alfredii [59] than in
non-hyperaccumulating relatives. A lower sequestration into root
vacuoles accounts also for the enhanced As translocation in hyperaccumulator compared with non-hyperaccumulator species of
Pteris [52].
Constitutively large quantities of small organic molecules are
present in hyperaccumulator roots that can operate as metalbinding ligands. However, the involvement of different chelators
in hyperaccumulation strategies has not been quite established
yet. The role of organic acids, mainly malate and citrate, as lig-
ands in the root cells is particularly controversial, due to their low
association constants with metals that makes complexation negligible at cytosolic pH values. They may be relevant only within
the acidic vacuolar environment [60]. A key role in heavy metal
hyperaccumulation seems to be played by free amino acids, such
as histidine and nicotinamine, which form stable complexes with
bivalent cations [61]. Free histidine (His) is regarded as the most
important ligand involved in Ni hyperaccumulation [61]. In roots
of the Ni hyperaccumulator Alyssum lesbiacum, as compared with
the non-hyperaccumulator A. montanum, the constitutive overexpression of the TP-PRT1 gene (encoding the ATP-phosphoribosyl
transferase enzyme committed in the first step of the biosynthetic pathway) leads to a larger endogenous pool of His, which
favours the Ni xylem loading as a Ni-His complex [62,63]. The
high concentrations of His in roots of different Ni hyperaccumulating Thlaspi species suggests that the amino acid may operate
in the same way in other hyperaccumulators [31]. Moreover, in
hyperaccumulators, but not in non-hyperaccumulators, the Ni–His
complexation, besides the involvement in sustaining the Ni release
into the xylem, plays an essential role in preventing the heavy metal
entrapment in root cell vacuoles, thus keeping it in the cytosol, in a
detoxified form available for translocation [23,64]. Genes encoding
enzymes of the nicotinamine biosynthetic pathway are overexpressed in roots of the Zn/Ni hyperaccumulators T. caerulescens
and A. halleri which contain 3-fold higher amount of nicotinamine than roots of congener non-hyperaccumulating species
[43,65,66]. However, in T. caerulescens, enhanced nicotinamine
synthesis and nicotinamine–metal chelation show a positive correlation with Ni hyperaccumulation [67], whereas in A. halleri they
are involved in Zn hyperaccumulation, with a possible role for
the cytosolic nicotinamine–Zn complexes also in keeping metal
N. Rascio, F. Navari-Izzo / Plant Science 180 (2011) 169–181
ions in detoxified form disposable for loading into xylem vessels
[43,68].
A large body of evidence indicates that fast and efficient
root-to-shoot translocation of large amounts of heavy metals in
hyperaccumulator plants relies on enhanced xylem loading by
a constitutive overexpression of genes coding for transport systems common to non-hyperaccumulators. The P1B -type ATPases,
a class of proteins, also named HMAs (Heavy Metal transporting
ATPases), are of particular importance (Fig. 2). They operate in
heavy metal transport and play a role in metal homeostasis and
tolerance [69]. Genes encoding bivalent cation transporters belonging to HMAs (among which HMA4) are overexpressed in roots and
shoots of Zn/Cd hyperaccumulators T. caerulescens and A. halleri
[66,70–72]. Moreover, the HMA4 expression is up-regulated when
these plants are exposed to high levels of Cd and Zn, whereas
it is down-regulated in non-hyperaccumulator relatives [70]. The
overexpression of HMA4 supports a role of the HMA4 protein
(which belongs to the Zn/Co/Cd/Pb HMA subclass and is localized
at xylem parenchyma plasma membranes) in Cd and Zn efflux from
the root symplasm into the xylem vessels, necessary for shoot
hyperaccumulation. This role is upheld by QTL analysis showing
co-localization of a major QTL for Zn and Cd tolerance with the
HMA4 gene in A. halleri [73–75]. Interestingly, it has been demonstrated that the HMA4 activity positively affects other candidate
genes for hyperaccumulation. In fact, the increased expression of
HMA4 enhances the expression of genes belonging to the ZIP family,
implicated in heavy metal uptake. This strongly suggests that the
root-to-shoot translocation acts as a driving force of the hyperaccumulation, by creating a permanent metal deficiency response in
roots [72].
The MATE (Multidrug And Toxin Efflux) family of small organic
molecule transporters seems to be another kind of transport
proteins that are active in heavy metal translocation in hyperaccumulator plants (Fig. 2). FDR3, a gene encoding a member of this
family, is constitutively overexpressed in roots of T. caerulescens
and A. halleri [66,76]. The FDR3 protein, which is localized at root
pericycle plasma membranes, usually operates in the xylem influx
of citrate, which is required as a ligand for Fe homeostasis and transport [77], but its overexpression in hyperaccumulators suggests
that FDR3 might also play a role in translocation of other metals, such as Zn [78]. Moreover, evidence exists for the involvement
in heavy metal translocation by YSL (Yellow Strip1-Like) family
members (Fig. 2), which mediate the loading into and unloading
out of xylem of nicotinamine–metal chelates [79]. Three genes
(TcYSL3, TcYSL5 and YSL7), are constitutively overexpressed in roots
and shoots of T. caerulescens where the YLS proteins do participate in vascular loading and translocation of nicotinamine–metal
(especially nicotinamine–Ni) complexes [80]. The transport system involved in xylem loading of Ni–His complexes occurring in
hyperaccumulator roots, has not yet been elucidated. Comparative analyses between the Ni hyperaccumulator Thlaspi goesingense
and the non-hyperaccumulator T. arvense have revealed that, under
non-toxic conditions, both species display similar root-to-shoot
Ni transport rates [81]. The authors conclude that the hyperaccumulation ability of T. goesingense depends on a very efficient Ni
detoxification and/or sequestration mechanisms, much more than
on enhanced heavy metal translocation.
The greater arsenic translocation to the shoot in hyperaccumulator P. vittata, as compared with non-hyperaccumulator ferns,
occurs principally as arsenite, which accounts for over 90% of the
As in the xylem sap [82]. This is because in the roots of hyperaccumulating ferns most of arsenate (AsV ) is quickly reduced to
arsenite (AsIII ) by the activity of an exclusive glutathione dependent arsenate reductase [83]. The remaining arsenate can be loaded
into xylem by phosphate transporters, while the efflux toward
the vascular tissues of the predominant arsenite requires differ-
173
ent transport systems, which have yet to be identified. However,
some evidence supports aquaglyceroporins of the NIP (Nodulin
26-like Intrinsic Proteins) subfamily as the most likely candidates
(Fig. 2). These plasma membrane proteins, that specifically operate in arsenite transport in mammals [84], also mediate arsenite
transport in plants [85,86]. Thus a high expression of such proteins
might conceivably account for the arsenite transfer from root cell
cytoplasm to xylem vessels in As hyperaccumulators [87]. Most of
Se taken up by root cells of Se hyperaccumulators remains as selenate. Thus, its root-to-shoot translocation occurs through sulphate
transport systems [88] (Fig. 2).
Whether the long-distance xylem transport of heavy metals
can occur in free ionic forms or through metal complexation with
organic acids is still controversial. Most of Zn and Cd, for instance, is
present as free hydrated cations in the xylem sap of T. caerulescens
and A. halleri [89,90], and only one-third of Ni is bound to citrate
in the xylem of hyperaccumulator Stackhousia tryoni (Celastraceae)
[91]. Conversely, almost all Ni is complexed with citrate and other
organic acids in the latex of the extreme Ni hyperaccumulator S.
acuminata [92].
3.3. Detoxification/sequestration
Great efficiency in detoxification and sequestration is a key
property of hyperaccumulators which allows them to concentrate
huge amounts of heavy metals in above-ground organs without suffering any phytotoxic effect. This exceptionally high heavy metal
accumulation becomes even more astonishing bearing in mind
that it principally occurs in leaves where photosynthesis, essential for plant survival, is accomplished, and that the photosynthetic
apparatus is a major target for most of these contaminants. The
preferential heavy metal detoxification/sequestration do occur in
locations, such as epidermis [93–96], trichomes [97] and even
cuticle [98], where they do least damage to the photosynthetic
machinery. In many cases heavy metals are also excluded from both
subsidiary and guard cells of stomata [99–101]. This may preserve
the functional stomatal cells from metal phytotoxic effects.
The detoxifying/sequestering mechanisms in aerial organs of
hyperaccumulators consist mainly in heavy metal complexation
with ligands and/or in their removal from metabolically active
cytoplasm by moving them into inactive compartments, mainly
vacuoles and cell walls (Fig. 1). Comparative transcriptome analyses between hyperaccumulator and related non-hyperaccumulator
species have demonstrated that also the sequestration trait relies,
at least in part, on constitutive overexpression of genes that, in
this case, encode proteins operating in heavy metal transfer across
the tonoplast and/or plasma membrane and involved in excluding them from cytoplasm. CDF (Cation Diffusion Facilitator) family
members, also named MTPs (Metal Transporter Proteins), which
mediate bivalent cation efflux from the cytosol, are important
candidates (Fig. 2). MTP1, a gene encoding a protein localized at
tonoplast, is highly overexpressed in leaves of Zn/Ni hyperaccumulators [102–105]. It has been suggested that MTP1, besides the
role in Zn tolerance, may also play a role in enhancing Zn accumulation. The Zn transport into the vacuole, in fact, may initiate a
systemic Zn deficiency response that includes the enhancement of
the heavy metal uptake and translocation via the increased expression of ZIP transporters in hyperaccumulator plants [105]. MTP
members also mediate the Ni vacuolar storage in T. goesingense
shoots [106]. Moreover, the finding that MTP1 is localized at both
vacuolar and plasma membrane suggests that it can also operate in
Zn and Ni efflux from cytoplasm to cell wall [103].
The overexpression of HMA3, coding for a vacuolar P1B -ATPase,
plausibly involved in Zn compartmentation, and that of CAX
genes encoding members of a cation exchanger family that seems
to mediate Cd sequestration (Fig. 2), have been noticed in T.
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caerulescens and A. halleri and supposed to be involved in heavy
metal hyperaccumulation [107,108].
Storage of As as inorganic arsenite in vacuoles is a key mechanism found in fronds of hyperaccumulator ferns, although the
transport system located at the tonoplast has not been identified
yet [87].
Small ligands, such as organic acids, have a major role as detoxifying factors. Such ligands may be instrumental in preventing
the persistence of heavy metals as free ions in the cytoplasm
and even more in enabling their entrapment in vacuoles where
the metal–organic acid chelates are primarily located. Citrate, for
instance, is the main ligand of Ni in leaves of T. goesingense [109],
while citrate and acetate bind Cd in leaves of S. nigrum [16]. Moreover, most Zn in A. halleri and Cd in T. caerulescens are complexed
with malate [89,110].
The heavy metal detoxification in hyperaccumulators, in contrast with tolerant non-hyperaccumulator plants, does not rely on
high molecular mass ligands, such as phytochelatins [111,112],
likely because of the excessive sulphur amounts and the prohibitive
metabolic cost that a massive synthesis of this kind of chelators would require [113]. Overexpression of antioxidation-related
genes [114], as well as enhanced synthesis of glutathione (GSH) as
pivotal antioxidant molecule [108], do occur, instead, in hyperaccumulators, as a strategy to reinforce the cell antioxidant system
and cope with the risk of ROS rise due to heavy metal stress.
The major detoxification strategy in Se hyperaccumulators
is to get rid of selenoaminoacids, mainly selenocysteine (SeCys), derived from selenate assimilation in leaf chloroplasts.
Selenoaminoacids are misincorporated in proteins instead of sulphur amino acids, resulting in Se toxicity. This detoxification occurs
through methylation of Se-Cys to the harmless non-protein amino
acid methylselenocysteine in a reaction catalyzed by a selenocysteine methyltransferase, which is constitutively expressed and
activated only in leaves of hyperaccumulator species [115].
4. Why did plants evolve hyperaccumulation of heavy
metals?
The discovery of a class of plants that concentrate exceptionally
high amounts of normally toxic heavy metals in leaves has attracted
considerable interest, and challenged biologists to find reasons for
this unusual behaviour by providing answers to the question: why
do some plants do it? In other words: what functions does hyperaccumulation perform in these plants and what are the benefits and
the adaptive values of metal hyperaccumulation?
A variety of hypotheses have been proposed to explain the role
of high elemental concentrations in leaves [116], namely: metal
tolerance/disposal, drought resistance, interference with neighbouring plants, and defence against natural enemies. According to
the tolerance/disposal hypothesis, the peculiar hyperaccumulation
pattern would allow plants to take heavy metals away from the
roots by sequestering them in tolerant leaf tissues. This eliminates
them from the plant body by shedding the high-metal aerial organ.
Another postulated explanation is that large amounts of heavy
metals might increase plant drought resistance, with a waterconserving role in the cell walls or acting as osmolytes inside
the cells. These hypotheses, however, are hardly supported by
experimental evidence, so that their validation deserves further
investigation. The interference hypothesis, also termed “elemental allelopathy”, suggests, instead, that perennial hyperaccumulator
plants may interfere with neighbouring plants through enrichment
of metal in the surface soil under their canopies. This gives rise to a
high-metal leaf litter that prevents the establishment of less metal
tolerant species. One group has measured higher Ni levels in the
surface soil under the canopy of hyperaccumulator S. acuminata
than under that of non-hyperaccumulator species [117]. However,
another group has questioned elemental allelopathy finding that
high-Ni leaves shed by Alyssum murale do not create a “toxic zone”
around the Ni hyperaccumulator and do not inhibit seed germination of competing plants [118]. The lack of allelopathic effect is
probably due to the fact that most Ni released from the leaf biomass
does not remain in a soluble and phytoavailable form, but is rapidly
bound to soil constituents thus becoming unable to affect neighbouring plants. Also the hypothesis of elemental allelopathy does
not have satisfactory experimental verification yet [119].
4.1. The “elemental defence” hypothesis
The hypothesis which has attracted most attention suggests
that the high heavy metal concentrations in aerial tissues may
function as a self-defence strategy evolved in hyperaccumulator
plants against some natural enemies, such as herbivores (Fig. 1)
and pathogens. This “elemental defence” hypothesis has been
widely tested, gaining much supporting evidence, although some
tests have led to contradictory responses. Some recent studies, for
instance, confirm the defensive function of Ni [120], Cd [121], Zn
[122], As [123] and Se [124] while others [125,126] seem to counter
the heavy metal involvement in plant defence. Despite the numerous reports regarding this popular hypothesis, more information is
required since few taxa have been tested, the majority of studies
have focused on Brassicaceae and only some elements (Ni, Zn, Cd,
As, Se) have been examined. Moreover, the defensive effects have
been analyzed mostly in laboratory conditions and have considered
only one or a few selected herbivores, rather than being tested in
the field where hyperaccumulators have to face an array of natural enemies [127]. The mix of contrasting conclusions reported in
literature about the effectiveness of heavy metals as defence elements might depend on different experimental conditions or heavy
metal concentrations used in each study, as well as on the ability
of certain herbivores to overcome the plant defence [127]. Heavy
metals, actually, may provide protection against a broad range of
enemies that the plant encounters in natural situations, whereas
some others may be able to feed on a hyperaccumulator species
despite its elemental composition. Mechanisms enabling herbivores to circumvent the heavy metal defence might be “avoidance”
which leads an herbivore to selectively eat only low-metal tissues of the plant and “dietary dilution”, which consists of lowering
overall metal ingestion by eating both high-metal and low-metal
tissues [127]. Another mechanism deserving a particular interest
is “tolerance”, in which physiological adaptations allow specialist
herbivores to withstand a high-metal diet, thus disarming the elemental defence of the plant [128,129]. The bug Melanotrichus boydi,
for instance, prefers to feed on the Ni hyperaccumulator Streptanthus polygaloides [130] and a strain of the moth Plutella xylostella
feeds on the Se hyperaccumulator S. pinnata without suffering from
the high-Se diet [131].
Heavy metals can act against herbivore through their toxicity,
but this does not safeguard the plant from undergoing damage
before poisoning the enemy. Thus a more effective defence from
herbivore attack should be through feeding deterrence. Experimental evidence exists that some herbivores prefer to eat low-Zn T.
caerulescens [132] and low-Ni Senecio coronatus [133] when offered
a choice between plants containing either low or high-metal concentrations. Deterrent effects have also been shown for Cd [121],
As [123] and Se [134]. This ability to avoid feeding on plants with
high heavy metal levels might support the view that herbivores
have a “taste for metals”, although no information exists on how
they might do it. However, since the metal treatment will strongly
affect the plants’ metabolome, it might be that herbivores do not
directly perceive metals in their food, but rather metal-induced
metabolites.
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175
4.2. The “joint effects” hypothesis
Chemical defence of plants from enemy attack can also involve
a variety of organic (secondary metabolite) compounds. However,
elemental defence offers some advantages over organic defence
[127]: the toxic elements are not synthesized by the plant but taken
up from the soil thus making the elemental defence less metabolically expensive than the organic one; the inorganic elements
cannot be biochemically degraded by most herbivores (although
some specialistic herbivores can chelate or sequester them), so
that this counter defence mechanism of the enemies is prevented.
The rather high cost of secondary metabolite biosynthesis accounts
for a “trade off” hypothesis, in which the metal-based defence
of hyperaccumulator plants may have evolved to reduce levels
of energy-demanding organic defences. Lower levels of defensive glucosinolates found in Ni hyperaccumulator S. polygaloides
[135] and in Zn hyperaccumulator T. caerulescens [136], when compared with congener non-hyperaccumulator species, support the
view of a trade off between metal hyperaccumulation and secondary metabolite synthesis. However, the trade off hypothesis
remains somewhat controversial. Differences in concentration of
specific glucosinolates, but not in their total amount between Ni
hyperaccumulating and non-hyperaccumulating plants, have been
measured [120], and it has been suggested that glucosinolates
rather than Zn are involved as deterrents in antiherbivore defences
of T. caerulescens populations [136].
Joint defensive effects may actually exist between elemental and
organic plant compounds, which may act in concert with each other
and enhance plant defence overall [127]. Heavy metal and several
defensive organic metabolites operate additively against an herbivore enemy in Ni hyperaccumulator S. polygaloides [137]. This
new joint effects hypothesis may justify the simultaneous presence
of elemental and organic defences as well as the hyperaccumulation of more than one heavy metal in the same plant. Joint effects
between heavy metals, besides that between a heavy metal and
an organic chemical, have been highlighted [137]. However also
this new interesting idea of a defensive enhancement achieved in
hyperaccumulators through joint effects of elemental and organic
plant defences needs to be supported by future investigation.
5. Why do hyperaccumulators attract so much interest?
Besides their ecological and physiological interest, hyperaccumulator plants have received considerable attention due to the
possibility of exploiting their accumulation traits for practical
applications, in particular to develop technologies for phytoremediation of heavy metal contaminated soils or for mining valuable
metals from mineralized sites.
5.1. Potential application in phytoremediation
The last two decades have seen the emergence of eco-friendly
soil remediation techniques, collectively known as phytoremediation, that utilize plant species. The use of plants to remediate
polluted soils is seen as having great promise compared to
conventional, civil-engineering methods and several recent comprehensive reviews summarising the most important aspects of
soil metal phytoremediation are available [14,138–142]. The naturally occurring heavy metal hyperaccumulator plants which, when
growing in metal-enriched habitats, can accumulate 100–1000fold higher levels of metals than normal plants are excellent
candidates for phytoextraction (Fig. 3), as these plants take up from
the soil two or three orders of magnitude more metals than plant
species growing on uncontaminated soils.
Chaney et al. [143] are the first to have proposed the exploitation of heavy metal hyperaccumulator plants to clean up polluted
Fig. 3. Phytoremediation and phytomining of heavy metals rich soils by using plants
which hyperaccumulate these metals in above-ground organs. The harvesting of the
aerial part of the plants leads to the disposal of the huge amounts of toxic heavy
metals removed from the soil or to the recovery of the valuable metals taken up.
sites. However, hyperaccumulators have been later believed to
have limited potential of phytoremediation because most of them
are metal selective, have not been found for all elements of interest, can be used in their natural habitats only, and, above all, have
small biomass, shallow root systems and slow growth rates, which
limit the speed of metal removal [144,145]. In addition, there is
no knowledge about the agronomics, genetics, breeding potential,
and disease spectrum of these plants. This is the case for many
hyperaccumulator plants including the Zn/Cd hyperaccumulator T.
caerulescens, which gives a maximum of 2 tons ha−1 of shoot dry
matter. Although the annual biomass yield is an important trait
for phytoremediation, the ability to hyperaccumulate and hypertolerate metals is of greater importance than high biomass [14].
Pot and field studies have shown that the hyperaccumulator T.
caerulescens grown as a crop can attain as high as 5 tons ha−1 by
breeding to increase the combination of yield and shoot metal concentration [146]. Furthermore, the recycling of shoot metals may
provide added value to the ash from metal hyperaccumulators, so
that there is no need to pay for disposal of the plants. Various
species of Thlaspi are known to hyperaccumulate more than one
metal. Predominantly, Thlaspi grows on Ni contaminated sites and
accumulates about 3% of its dry matter as metal but T. caerulescens
can accumulate Cd, Ni, Zn and also Pb. As a hyperaccumulator of Cd
and Zn it could remove as much as 60 kg Zn ha−1 and 8.4 Kg Cd ha−1
[147]; T. goesingese and T. ochroleucum hyperaccumulate Ni and
Zn while T. rotundifolium hyperaccumulates Ni, Pb and Zn [148].
The brake fern P. vittata, which produces a relatively large biomass
under favourable climate conditions, accumulates (from relatively
low As concentration in the soil) 22 g As kg−1 in the frond dry
weight, with a bioconcentration factor of 87 and a removal of 26%
of the soil’s initial As ([29,139] and literature cited therein). These
results suggest that phytoremediation of at least moderately Ascontaminated sites is feasible. Although Pb is largely immobile in
soil and its extraction rate is limited by solubility and diffusion
to the root surface, common buckwheat (Fagopyrum esculentum,
Polygonaceae), the first known Pb hyperaccumulator species with
high biomass, can accumulate up to 4.2 mg g−1 dry weight of Pb in
the shoots [149]. Amending the soil with the biodegradable methylglycine diacetic acid (MGDA) resulted in a 5-fold increase in the
Pb shoot concentration. This relevant finding qualifies this species
as an excellent candidate for remediating Pb-contaminated soils.
Phytolacca acinosa (Phytolaccaceae), a plant that grows rapidly and
has substantial biomass, has been considered to have potential for
use in phytoremediation. The plant can accumulate 19.3 g Mn kg−1
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dry weight when grown on Mn-rich soils [150]. The efficiency of
Alyssum serpyllifolium subsp. lusitanicum for use in phytoextraction of polymetal-contaminated soils has been examined [151].
The plants have been grown on soils contaminated with Cr, Cu,
Pb and Zn. The results suggest that A. serpyllifolium can be suitable
for phytoextraction in polymetal-polluted soils, provided that Cu
concentrations are not phytotoxic. However, with the hyperaccumulators available, decades are needed to clean up contaminated
sites. It has been calculated that to decrease Zn concentration from
440 to 300 mg Zn kg−1 soil nine croppings of T. caerulescens would
be required [152] and 28 years of cultivation of this plant would be
needed to remove 2100 mg Zn kg−1 from a soil [153]. T. caerulescens
is useful for moderately Zn- and Cd-contaminated soils but would
take far too long on highly contaminated ones. It also appears that
season length, method of sowing seeds and soil pH have effects on
the Zn and Cd extraction capacity of T. caerulescens from the soil
[154,155]. The efficiency of phytoextraction, besides biomass production, depends on the metal bioconcentration factor (plant to soil
concentration ratio) and for Zn and Cd it decreases log-linearly with
soil metal concentration [156]. Moreover, the phytoremediation
potential differs between different population of T. caerulescens.
The southern French ecotype showed a higher ability to accumulate Cd than Zn: the different uptake of Cd and Zn shows that there
are basic differences in the mechanism of accumulation of both
metals in hyperaccumulators [45,154]. Thus, increased selection for
traits of interest may help to improve the phytoremediation capacity of hyperaccumulators. A crop of T. caerulescens or A. halleri could,
after cropping, remove decades-worth of Cd accumulation from
pastures that have been treated with Cd-rich phosphate fertilizers
[157]. Small-scale field experiments have also been conducted with
Alyssum bertolonii and Berkheya coddii, fast-growing Ni hyperaccumulators [158,159]. The combination of high biomass and high-Ni
content, together with its easy propagation and culture as well as
its tolerance to cool climate conditions, should render B. coddii suitable for Ni-phytoremediation. For moderate Ni contamination two
crops of B. coddii would be sufficient to reduce the metal concentration to well below the EU guidelines and for A. bertolonii, which has
a lower biomass, from 5 to 10 annual crops would be needed. A 2year field study has been conducted to determine the efficiency
of the fern P. vittata on As removal at an As-contaminated site.
Approximately 19.3 g of As have been removed from the soil and
it has been estimated that 8 years would be needed to completely
remediate the soil in order to meet the residential site and/or commercial site requirements [160,161]. However, some estimates are
based on achieving a soil clean up goal of 40 mg As kg−1 from an
average of 82 mg As kg−1 [161], while that of others [160] contains on average 190 mg kg−1 . The presence of Pb in one soil [161]
may have hindered the ability of P. vittata from removing As from
it.
It has been demonstrated that T. caerulescens, although being
able to mine the soil metals more efficiently than non-accumulator
plants, exhibits the same capacity of non-accumulators to increase
metal availability in the rhizosphere, so that the use of amendments to raise metal solubility has been suggested. A number of
possible amendments such as ethylene diamine tetracetic acid
(EDTA), nitrilotriacetic acid (NTA) and citric acid have been tested
under field conditions and seem to have no effect on increasing metal content, but may actually decrease biomass production
of hyperaccumulators, thus reducing their potential of phytoextraction [139,154,158,159,162]. Recently, the effects of polycyclic
aromatic hydrocarbons (PAHs) on the extractability of Ni by A. lesbiacum have been investigated. Plant growth is negatively affected
by PAHs; however, there is no significant effect on the phytoextraction of Ni per unit biomass of shoot, indicating that A. lesbiacum
might be effective in phytoextracting Ni from marginally PAHcontaminated soils [163]. No hyperaccumulators of radionuclides
have been reported so far, thus it is unlikely that the hyperaccumulation strategy is possible for these contaminants
In summary, despite intensive research very few field studies
or commercial operations that demonstrate successful phytoextraction by hyperaccumulators have been realized, so it could be
considered a valuable tool only for Ni and As, while for the other
metals the technology still appears to be far from the practice. At
the moment phytoextraction with hyperaccumulators is an option
to decontaminate soils with low to moderate metal concentrations
while for highly contaminated soils it should be considered as a
long-term remediation process.
5.2. Transfer of hyperaccumulation traits to rapidly growing
species
A promising biotechnological approach for enhancing the
potential for metal phytoextraction, may be to improve the hyperaccumulator growth rate through selective breeding, or by the
transfer of metal hyperaccumulation genes to high biomass species.
In an effort to correct for small sizes of hyperaccumulator plants,
somatic hybrids have been generated between T. caerulescens and
Brassica napus. High biomas hybrid selected for Zn tolerance are
capable of accumulating Zn level that would have been toxic to B.
napus [164] indicating that the transfer of the metal hyperaccumulating phenotype is feasible. Somatic hybrids from T. caerulescens
and B. juncea are also able to remove significant amounts of Pb
[165]. T. caerulescens has also been used as source of genes for developing plant species better suited for phytoremediation of metal
contaminated soils [166].
Bioengineered plants tolerant to the presence of toxic levels of
metals like Cd [167], Zn, Cr, Cu, Pb [168], As [169] and Se [170] have
been reported. A combination of transporter genes has also been
used in rapidly growing plant species leading to promising results
[169,171–173]. Transgenic B. juncea, grown either in hydroponic or
in soils, shows higher uptake of Se and enhanced Se tolerance than
the wild species [174,175]. To engineer Se tolerance the selenocysteine methyltransferase (SMT) gene has been transferred from
the Se hyperaccumulator A. bisulcatus to Se-non-tolerant B. juncea.
SMT transgenic plants of B. juncea grown in a contaminated soil
accumulate 60% more Se than the wild-type ([176] and literature
reported therein).
The transgenic plant approach has shown to be promising, but
only very few studies have been performed till now under field
conditions [176]. Moreover, it has to be considered that tolerance
and accumulation of heavy metals and thus phytoextraction potential of a given plant are controlled by many genes, so that genetic
manipulations to improve these traits in fast-growing plants will
require to change the expression levels in a number of genes, and
to cross them to determine the number of genes involved and
their characteristics. Functions and regulations of genes involved
in metal hyperaccumulation, uptake, root-to-shoot translocation,
detoxification/sequestration mechanisms need to be fully understood to render transgenic approach not far to solve the problem.
5.3. Potential application in phytomining
Phytomining (a subset of phytoextraction) aims to generate
revenue by recovering marketable amounts of metals from plant
biomass (bio-ores) [177] through the use of plants to mine valuable
heavy metals from contaminated or mineralized soils (Fig. 3).
A pioneer phytomining study has been carried out using the
Ni hyperaccumulator S. polygaloides [178]: a yield of 100 kg ha−1
of sulphur-free Ni could be obtained after moderate application of
fertilizers. The removal of Ni from soil using phytomining is viable
in principle, since there are many hyperaccumulator plants, such as
Alyssum spp. and B. coddii, fulfilling the criterion of achieving shoot
Ni concentrations higher than 10 g kg−1 on a dry matter basis and
N. Rascio, F. Navari-Izzo / Plant Science 180 (2011) 169–181
producing more than 10,000 kg ha −1 per year [177]. A. bertolonii
can also accumulate 10 mg Ni g−1 dry matter from serpentine soils
[179]. Experiments have been carried out on the potential use of
this hyperaccumulator plant in phytomining of serpentine soils.
In the field trial plants of A. bertolonii have been fertilized with
N + P + K over a period of 2 years. Fertilization increases biomass
3-fold without dilution of Ni concentration in the fertilized plants.
It has been concluded that A. bertolonii, with a biomass after fertilization of about 13,500 kg ha−1 , or other species of Alyssum might
be used for phytomining [158]. In another field trial B. coddii, with
an unfertilized biomass of 12,000 kg ha−1 , has been reported as one
of the best candidates for phytomining of Ni with applied fertilizers and adequate moisture, after which a biomass of 22,000 kg ha−1
and a high-Ni concentration has been achieved [159,162,177,180].
The potential of this species for phytomining has also been evaluated and a yield of 100 kg ha−1 of Ni should be achievable at
many sites worldwide [180]. On the basis of biomass, the highNi concentration in the harvestable parts of the plants and the
additional money obtained from the energy of combustion either
of the Ni hyperaccumulator S. polygaloides or A. bertolonii, it has
been concluded that the return to a farmer growing a “crop of
nickel” would be comparable, or even superior, to that obtained
for a crop of wheat [158,181]. Furthermore, if the above plants
are used for phytoremediation of Ni polluted soils as a result of
industrial activity, it would surely be of economic benefit considering the very high costs of conventional extraction methods and of
storage of the toxic materials. Commercial phytomining technologies employing Alyssum Ni hyperaccumulator species have been
developed [100]. However, hyperaccumulator plants might realistically also be expected to be used for Au, Tl, Co and U as well.
Each has a high world price for the target metal and plants might
extract from soils or mine tailing containing concentrations of the
metals at a level uneconomic for conventional extraction techniques. Iberis intermedia and Biscutella (Brassicaceae) have been
proposed for phytomining of Tl [180]. For other less valuable metals
(Pb, Cd, 137 Cs, Cu, Se) phytomining will never emerge as a profitable agricultural industry. Notwithstanding all these promising
field studies and the reported advantages over conventional mining [162], up to now there is no report of successful commercial
phytomining operations. The potential limiting factors to the commercialization of phytomining have been investigated [182] and it
has been concluded that is only attractive when applied to a contaminated site and might be usefully combined with conventional
mining. In conclusion, phytomining with high-biomass hyperaccumulators could have economic advantages over traditional mining
techniques, especially in cases where the extracted metals are biomining targets, have economic value and the energy of combustion
of biomass can be sold. In addition, as bio-ore is practically sulphurfree its smelting does not contribute to acid rain. At the moment
there is need to develop methods to recover and market the metals. Despite the large number of hyperaccumulators found to date,
there is insufficient information on the distribution of these species
or their uptake mechanisms so that they can be properly utilized in
phytomining. Neither are their agronomical properties, such as fertilizer requirements, soil pH management, weed control and water
requirements, adequately known. Notwithstanding these limitations it is clear that the commercialization of phytomining using
high-biomass hyperaccumulator plants depends essentially on the
metal concentration of the plant, its annual biomass production and
the world price of the target metal.
6. Conclusions and future directions
The problem of heavy metal pollution is continuously worsening due to a series of human activities, leading to intensification of
177
the research dealing with the phytotoxicity of these contaminants
and with the mechanisms used by plants to counter their harmful
effects.
Great interest has been gained by the behaviour of hyperaccumulator plants growing on metalliferous soils, which accumulated
heavy metals in leaves at concentrations several 100-fold higher
than other plants. Aims of studying these heavy metal hyperaccumulator species has been to highlight physiological and molecular
mechanisms underlying the hyperaccumulation ability, to discover
the adaptive functions performed by hyperaccumulation in these
plants and to explore the possibility of using them as tools to
remove metals from contaminated or natural metal-rich soils.
However, in spite of important progress made in recent years by
the numerous studies accomplished, the complexity of hyperaccumulation is far from being understood and several aspects of this
astonishing feature still await explanation.
Hyperaccumulator plants, which are widespread on metal soils
in both tropical and temperate zones of all the continents, belong to
several unrelated families. This shows that the hyperaccumulation
capability has been evolved more than once, although its adaptive
value is still under debate. The recent idea that heavy metals would
provide an elemental defence to the plant through joint effects with
organic defence compounds requires much experimental investigation. More elements and a larger number of hyperaccumulator
species need to be examined to validate the hypothesis of defensive effects of heavy metals. Furthermore, the investigations need
to move from laboratory to field settings to provide realistic information about elemental defences in natural environments, where
a plant can be exposed to a plethora of herbivores with different
feeding modes, as well as to pathogens and parasites.
Considerable attention has been given to the possibility of
using hyperaccumulators for phytoremediation/phytomining of
contaminated or natural metal-rich soils. However, more extensive research under field conditions for longer durations is required
taking also into account that a specific phytoextraction prescription, due to the different site-specific conditions, cannot be
applied to every site, even if with the same chemical composition. It is of pivotal importance to increase the understanding
of hyperaccumulator-based remedial mechanisms because they
will be able to provide clues for optimizing the effectiveness of
phytoextraction with appropriate agronomic practices. In addition, knowledge acquired on genes involved in hyperaccumulation
mechanisms will open the opportunity to use biotechnology to
transfer specific genes to high-biomass promising species. Moreover, much research is still needed on rhizosphere and soil
microbial composition under field conditions, in order to identify
micro-organisms associated with metal solubility or precipitation. There is also an urgent need to find and characterize other
hyperaccumulators, to cultivate them and better assess agronomic
practices and management to enhance plant growth and metal
uptake by selective breeding and gene manipulation. Even then,
metal uptake might pose environmental risks, unless the biomass
produced during the phytoremediation process could be rendered
economical by burning it to produce bio-ore or converting it into
bioenergy. However it is only matter of time before the commercialization of phytoextraction using high-biomass hyperaccumulator
plants becomes widespread, considering that not only will it remediate contaminated sites but will generate income from agricultural
lands otherwise not utilized.
Last but not least, it has to be pointed out the interest in
the potential exploiting of hyperaccumulators as a rich genetic
resource to develop engineered plants with enhanced nutritional
value for improving public health [183] or for contending with
widespread mineral deficiencies in human vegetarian diets [184].
The strategies of food crop biofortification are still in infancy; however their paramount importance for the world’s population makes
178
N. Rascio, F. Navari-Izzo / Plant Science 180 (2011) 169–181
this an exciting line of future research in the field of essential elements hyperaccumulation.
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