Accepted Manuscript

Title: Functional biology of halophytes in thephytoremediation of heavy metal contaminated soils

Author: Michael James Van Oosten Albino Maggio

PII: S0098-8472(14)00272-XDOI: http://dx.doi.org/doi:10.1016/j.envexpbot.2014.11.010Reference: EEB 2888

To appear in: Environmental and Experimental Botany

Received date: 9-9-2014Revised date: 23-10-2014Accepted date: 19-11-2014

Please cite this article as: Oosten, Michael James Van, Maggio,Albino, Functional biology of halophytes in the phytoremediation ofheavy metal contaminated soils.Environmental and Experimental Botanyhttp://dx.doi.org/10.1016/j.envexpbot.2014.11.010

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Functional biology of halophytes in the phytoremediation of heavy metal contaminated soils

Michael James Van Oosten, Albino Maggio*almaggio@unina.it

Department of Agricultural Science, University of Naples “Federico II”, Via Università 100, C

Portici, Italy

HIGHLIGHTS

Halophytes are important plants for both saline agriculture and phytoremediation.

Halophyte adaptive mechanisms may confer tolerance to ions beyond sodium and chloride.

Further research should explore the relationship salt/metal tolerance in these species.

Abstract

Halophytic plants are characterized by their ability to survive, even thrive, at concentrations of

sodium and chloride ions that would be toxic to most crop species. Given the diminishing

prospects for the availability of fresh water for agriculture, halophytes represent an important

resource for both our understanding of the fundamental physiological mechanisms in salt stress

adaptation and utilization of saline waters for agriculture. Mechanisms of adaptation that allow

halophytes to survive high salt concentrations may be not exclusive to sodium and chloride and

may confer tolerance to other toxic ions, including the loosely defined family of heavy metals. It

has been recently shown that a number of these halophytes do indeed have ability to

accumulate heavy metals or tolerate high levels of toxic ions in the environment. These abilities

make some halophytes excellent candidates for phytoextraction and phytostabilization of heavy

metals in contaminated soils. This review addresses the general deleterious effects of heavy

metals in plants, present known mechanisms of adaptation to heavy metal stress in halophytes

and discusses the potential of halophytes for phytoremediation of contaminated soils.

Considering the multifaceted potential of halophytes for biomass production in marginal and/or

extreme environments, their potential role in the broader context of agriculture and food

security should be further explored.

Keywords: Halophytes, phytoremediation, phytoextraction, phytostabilization, phytoexcretion,

heavy metals, salt tolerance.

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1. Introduction

Halophytic plants represent a tremendous resource in biology to study the mechanisms of

adaptation and evolution to/in hyperosmotic environments. Halophytes have evolved to

adapt to harsh conditions such as high salinity, xerothermic environments, and cold

seasonal temperatures (Flowers et al., 2010) and they typically tolerate the presence of

toxic ions, mainly in the form of sodium and chloride (Flowers and Colmer, 2008).

Sensitivity and tolerance can vary greatly within a single genus where optimal

concentrations for growth and development may range from 20 to 500mM (Cheeseman,

2013). Significant advances have been made in understanding how halophytes have

adapted to high salinity (Flowers et al., 2010; Cheesman, 2013; Bressan et al., 2013).

Moreover, halophytes have received particular attention in past few years not just as

model species in salt tolerance research, but as potential forage, fibre, and biomass crops

as well as platforms for developing crop systems that use saline water and/or ameliorate

salinized soils (Abdelly et al., 2006; Fedoroff et al., 2010; Shabala, 2013). It is estimated

that 20% of all agricultural land and 50% of cropland throughout the world is salt

affected (Flowers and Yeo, 1995; Shabala, 2012). In addition, as water availability for

agricultural uses becomes limited, utilization of semi-saline and saline waters becomes an

alternative source of water but soil salinization remains a potential risk (Letey et al.,

2011). The application of halophytes that bioaccumulate Na+ and Cl- presents a means of

reducing salt content in soil where leaching and mechanical extraction is not economical

(Qadir et al., 2003; 2005). Sodium bioaccumulating species can be grown on saline soils

and harvested on a regular basis. Addition of water and fertilizer can accelerate the

bioremediation process (Keiffer and Ungar, 2002). Several studies on the use of

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halophytes in soil desalination have been reported in the literature. The obligate

halophyte, Sesuvium portulacastrum L., for example, proved successful in desalinating

soils with the potential to remove up to 1 t Na+ ha-1 through sequestration in roots and

shoots (Rabhi et al., 2008; Rabhi et al., 2010). Also Suaeda maritima and Sesuvium

portulacastrum have been shown to bioaccumulate salts in their tissues and reduce soil

salinity (Ravindran et al., 2007; Rabhi et al 2009). Similarly, the halophyte Sulla carnosa

Desf. demonstrated the ability to desalinize soils moderately, 0.3 t Na+ ha−1, and showed

potential as a forage crop (Jlassi et al., 2013). Deep rooted halophytes like Atriplex

lentiformis have been used to absorb salt from mildly saline effluent from waste-water

treatment facilities with the intent to protect aquifers in urban environments (Glenn et al.,

2009).

Despite these and further examples on the practical use of halophytes to recover

salinized soils/waters, their ability to withstand other adverse conditions and/or adapt to

agriculture-unfavourable or extreme environments at large has been extensively explored.

In some cases, halophyte adaptive mechanisms confer tolerance to other ions beyond

sodium and chloride. Evidence suggests that the evolutionary adaptations present in

halophytes can also confer tolerance to other toxic elements (Flowers et al., 2010).

Therefore, the enormous potential halophytes present to plant biology and agriculture is

not just limited to salt tolerance. As momentum builds within the research community

studying halophytes and salt tolerance, understanding how these species interact with

other abiotic stresses will become more important (Huchzermeyer and Flowers, 2013;

Wang et al., 2014). Many of the physiological and molecular mechanisms that contribute

to salt tolerance in halophytes, including the ability to limit entry of ions into the

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transpiration stream, ions compartmentalization, synthesis of organic solutes (Flowers

and Colmer, 2008; Manousaki and Kalogerakis, 2011) and a robust antioxidative system

(Freeman et al., 2006) are also found in heavy metal tolerant species. Therefore, it has

been proposed that heavy metal plants and halophytes share a number of processes in

common (Shevyakova et al., 2003).

In this review we summarize the main features of halophytic plants in relationship

to their potential in phytoremediation of heavy metals contaminated soils. Although we

have predominantly focused on the dual tolerance of halophytes to NaCl and heavy

metals and possible interactions between underlying physiological and molecular

mechanisms, we also refer to glycophytes when necessary, since for this group of plants

heavy metals tolerance has been better documented. In the very last section of this

review, we attempted to contextualize the role of halophytes in three important

agricultural systems to demonstrate that further research on halophytes could ultimately

contribute to recover contaminated soils, improve food security and decrease competition

for arable land.

2. Heavy metals accumulation in plants and their environment

Heavy metals represent a significant environmental pollutant and can have serious effects

on soil and water quality, plant and animal nutrition, as well as human health

(Schwarzenbach et al., 2010; Jomova and Valko, 2011; Alloway, 2013). The generic

term of “heavy metals” refers to elements that demonstrate metallic properties (transition

metals, metalloids, lanthanides, and actinides), have a high specific gravity, 5.0 or

greater, and are toxic, even at low concentrations. The definition of heavy metals is

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somewhat ambiguous (Duffus et al., 2002) and efforts have been made to more

accurately define them as potentially toxic elements (PTE). The PTE most commonly

referred to as “heavy metals” are arsenic (As), silver (Ag), cadmium (Cd), cobalt (Co),

chromium (Cr), copper, (Cu) iron (Fe), mercury (Hg), manganese (Mn), molybdenum

(Mo), nickel (Ni), lead (Pb), and zinc (Zn) (Alloway, 2013). These contaminants are

persistent in the environment unlike organic molecules.

Beginning in the mid-19th century, rapid industrialization resulted in the steep

increase of heavy metal emissions into the environment. In the past few decades,

concerns in developed countries led to changes in environmental policies and a curtailing

of emissions. The removal of Pb from gasoline and paints, internal waste water

recycling, and industrial recovery systems being a few examples (Schwarzenbach et al.,

2010; Alloway, 2013). In addition to soil contamination, which can have effects for

decades or longer, uptake and accumulation in living organisms is of particular concern

as these elements can be carcinogenic or reach toxic concentrations (Alloway, 2013).

Many industrial processes use and subsequently produce heavy metal wastes. Waste and

by-products generated by mining sties, foundries, smelters, coal-fired power plants, metal

plating, tanneries, battery production, and the paper industry all have the capability of

contaminating the environment with heavy metals (Alloway, 2013).

The bulk properties of soils have been found to be good predictors of metal

toxicity to plants (Giller et al., 2009). Physical-chemical factors, such as cation exchange

capacity (CEC) and pH, influence heavy metal soil toxicity to plants (Rooney et al.,

2006), as well as biotic determinants, including the bioavailability and microbial

populations present in the soil (Giller et al., 2009). This explains the toxicity variants

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observed in different soils containing the same concentration of heavy metals. It has

been also demonstrated that sub-populations and microbial groups adapt over time to

high levels of heavy metals in the soil (Giller et al., 2009). The application of sewage

sludge, partially or wholly un-treated, a common agricultural practice in the developing

world, can be a source of contamination that gradually expose living organisms to

contamination and elicit their adaptation. This process is exacerbated by the urbanization

and industrialization which both produce sewage sludge that is often contaminated with

heavy metals (Dai et al., 2006; Signh and Agrawal, 2008). In recent decades, a number

of plant families and species have been found to be tolerant to high heavy metal

concentrations. Some of these species have been found at sites with naturally high levels

of heavy metals or on contaminated industrial sites. Some species are capable of

tolerance through exclusion mechanisms, yet others accumulate ions to levels that are

considered toxic to other species (Krämer, 2010; Rascio and Navari-Izzo, 2011). Plant

species able to accumulate heavy metals in their shoots at much higher levels than what is

present in the soil are often called “hyperaccumulating”. These tolerant species are often

found in naturally metal rich soils (Brooks, 1998).

3.1 How heavy metals damage or stress plants

The phytotoxicity of heavy metals is well established (Foy et al., 1978). Metal ions cause

toxicity through three processes: 1) generation of reactive oxygen species that challenge

antioxidant defences and cause oxidative stress; 2) direct interaction with proteins

through affinity to thioyl-, histidyl-, and carboxyl groups; 3) ability to displace essential

cations in specific binding sites (Sharma and Dietz, 2009).

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Redox active heavy metals like Fe, Cu, Cr, V and Co form OH• from H2O2 through

the Haber-Weiss and Fenton reactions. These radicals damage cells through non-specific

lipid peroxidation (Dietz et al., 1999; Sharma and Dietz, 2009). Lipid peroxidation by

ROS results in the formation of oxidized lipids that can interfere with protein function

through uncontrolled hydrophobic interactions (Farmer and Mueller, 2013).

For example, the application of cadmium to plant cells induces cell death through

generation of ROS in three waves; H2O2 NADPH oxidase dependent accumulation, O2−

formation in mitochondria and finally generation of hydroperoxy fatty acids (Garnier et

al., 2006; Møller). In addition, cadmium may directly disrupt photosynthesis by

inhibiting CO2 fixation and Photosystem II (Sandalio et al., 2012). Photoactivation of PS

II is inhibited by cadmium competing with calcium for essential binding sites in the salt

tolerant unicellular green alga Chlamydomonas reinhardtii (Faller et al., 2005). While

cadmium has specific effects on photosynthesis through competition, heavy metals in

general increase competition for metalloproteins and their native cofactors.

Metalloproteins can suffer from competition between heavy metals and their native metal

cofactor disrupting metalloprotein functions like enzyme catalysis, signal transduction,

and stabilization of DNA and proteins (Dudev and Lim, 2014).

3.2 Phytoremediation strategies

Phytoremediation has been classified by three basic strategies: 1) phytoextraction - the

removal on contaminants from the soil by plants, 2) phytostabilization - the

immobilization of contaminants to a confined area, 3) phytodegradation - the destruction

of organic pollutants by plants. Plants capable of phytodegradation can operate through

phytovolatilization, rhizodegradation or phytodegradation per se. Phytovolatilization is

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the process in which plants take up contaminants and release them into the atmosphere

through transpiration (Salt et al., 1998). Degradation of contaminants in the rhizosphere

by root exudate and microbes is referred to as rhizodegradation (Macková et al., 2006).

When these contaminants are taken up by the plant and degradation occurs through

metabolism the process is called phytodegradation (Gerhardt et al., 2009).

Most naturally metal-accumulating plants could be suitable candidates for

phytoremediation. However, these plants generally produce low amounts of annual

biomass and are not suitable for large scale soil detoxification. For these reasons high

biomass crops that accumulate moderate levels of metals in their shoots are

suggested for field scale phytoremediation, since a greater aboveground biomass yield

can more than compensate for the lower PTE concentration in plant tissues

(Fiorentino et al., 2013). However, many naturally metal-accumulating plants are

glycophytes and do not grow well in saline environments (Ben Rejeb et al., 2013).

4.1 Control of heavy metals uptake, mobilization and activity in tolerant species

Metal tolerant plant species utilize several adaptation mechanisms to regulate the uptake,

mobilization and activity of heavy metal ions in the cytosol (Hossain and Komatsu,

2013). Exposure to heavy metals also initiates signalling processes in which the primary

cell wall is thought to be the initial site of extracellular signalling (Dal Corso et al.,

2010). Heavy metal stress induces changes in calcium levels, kinase activity and

subsequent changes in gene expression. Many of these responses crosstalk with other

stresses (Thapa et al., 2012). Changes in gene expression permit the plant challenged by

toxic ions to initiate survival and adaptation strategies, including plasma membrane

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exclusion to protect the cytosol as well as chelation and sequestration in the vacuole.

Metal hyperaccumulating species have adapted a similar strategy for compartmentalizing

toxic ions by translocation to the vacuole or cell wall. In tissues and organelles where the

metals are accumulated they are chelated by organic acids; which facilitate

hyperaccumulation (Krämer, 2010). Consequently, high concentrations of organic acids

such as malate or citrate and other chelators are often found in hypertolerant or

hyperaccumulating species (Sun et al., 2011). The halophyte, Spartina maritima

demonstrated significantly enhanced heavy metal uptake when grown in conjunction with

low weight organic acids. Application of citric acid to this species increased Zn uptake

by 85% and acetic acid increased Ni concentration in roots by 139%. Moreover,

chromium uptake was dramatically increased by application of low molecular weight

organic acids, with acetic acid (1032%) and malic acid (770%) having the largest effects

(Duarte et al., 2011). Organic acids can complex with heavy metals in the cytosol to

protect cellular machinery (Hall, 2001; Duarte et al., 2011; Chai et al., 2012). The

transport and sequestration of chelated metals is a key strategy for removing non-

essential as well as excess essential heavy metals from the cytosol (Mendoza-Cózatl et

al., 2011). Metal detoxification through sequestration in the vacuoles of leaves is highly

effective. The mobility of these metals is enhanced by decreasing sequestration of heavy

metals in the vacuoles of roots (Krämer, 2010). Uptake, translocation and eventual

sequestration involve high expression levels of membrane transporters (Rascio and

Navari-Izzo, 2011). ABC transporters play a key role in As, Cd and Hg detoxification,

transporting phytochelated metals into the vacuole (Park et al., 2011). ABC transporters ACCEPTED M

ANUSCRIPT

have been identified as vacuolar phytochelatin transporters in S. pombe and A. thaliana,

facilitating sequestration of toxic heavy metals.

Critical cytosolic processes are protected from the deleterious effects of heavy

metal generation of ROS by production of ROS scavenging proteins and molecular

chaperones. The ROS scavenging proteins are particularly critical for plants under stress

to maintain redox homeostasis (Hossain et al., 2012; Dal Corso et al., 2013a). Sulphur

metabolism also plays a role in heavy metal detoxification. Heavy metal accumulators

often have increased cysteine biosynthesis induced by heavy metals (Dal Corso et al.,

2013b). Highly reactive heavy metals such as Cr, Cu, and Fe are directly involved in

redox reactions and generate ROS (Hossain and Komatsu, 2013) and redox inactive

heavy metals can induce indirect ROS formation through depletion of antioxidant pools

(Hossain et al., 2012). Mild Cd stress can be overcome through adaptation though severe

stress can harm the photosynthetic machinery and lead to a reduction in RuBisCo levels,

increased lipid peroxidation and disruption of photosystem I (Villiers et al., 2011).

Exposure to Cd in rice leads to depletion of glutathione levels and a global increase in

proteins with antioxidant properties, preferentially in the roots (Lee et al., 2010). In

contrast, ROS response mechanisms, CAT and SOD activities, as well as proline levels

were enhanced at high concentrations of Cd and Ni in the halophyte Salicornia brachiata,

which is capable of withstanding high levels of Cd, Ni and As and accumulating these

heavy metals in foliar tissue (Sharma et al., 2009).

Glutathione (GSH) is the precursor for phytochelatins' (PC) synthesis in A.

thaliana (Rouhier et al., 2008). Both GSH and PCs have a high affinity for heavy metals

such as Ni, Cd, Hg, As (Cobett and Goldsbrough, 2002). In A. thaliana, GSH S-

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conjugates can be sequestered in the vacuole and involved in removing potentially

damaging heavy metals from sensitive cellular compartments (Grzam et al., 2007;

Paulose et al., 2013). Glutathione plays a key role in nickel tolerance within the Thlaspi

nickel accumulating species (Freeman et al., 2004). These species have constitutively

elevated glutathione levels that correlate with their ability to tolerate high levels of nickel.

Mature Thlaspi goesingense plants typically have 412.5 ± 80 nmol g-1 fwt of GSH while

Arabidopsis thaliana was found to have 268.86 ± 48.1 nmol g-1 fwt (Freeman et al.,

2004). Overexpression of serine acetyltransferase in Arabidopsis thaliana, a non-tolerant

relative of Thlaspi goesingense, caused an increased accumulation of OAS, Cys, and

glutathione. The glutathione accumulation, as high as 496 ± 20 nmol g-1 fwt, increased

the resistance to oxidative stress and growth inhibition under nickel exposure as well as

Co and Cd (Freeman and Salt, 2007). These results indicate that extremophile species,

such as Thlaspi goesingense, can serve as resources to be screened for genes that can be

transferred to non-tolerant species. Changes in GSH production and localization in

response to salt stress have been well documented (Hasegawa et al., 2000; Rouhier et al.,

2008). Recent work with Arabidopsis has shown that mutants in GLUTATIONE S-

TRANSFERASE U17 have increased levels of ABA and GSH, 35% higher than wild-

type. These mutants with higher levels of GSH demonstrated increased tolerance to both

drought and salt stress through increased ABA accumulation and decreased stomatal

aperture (Chen et al., 2012). Production of GSH in halophytes and metal accumulators is

therefore a shared mechanism and a number of halophytes have been shown to have

elevated GSH levels and/or efficient enzymatic activity. Overexpression of the Salicornia

brachiata glutathione transferase in tobacco conferred tolerance to NaCl at

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concentrations up to 300 mM (Jha et al., 2011). Similarly, in the halophyte Sesuvium

portulacastrum (L.), antioxidants like GSH and ascorbate play a key role in balancing

redox relations under saline conditions (Lokhande et al., 2011).

Proteomic studies in metal hyperaccumulators have granted significant insight into the

proteins and networks responsible for accumulation and tolerance in selected species (Dal

Corso et al., 2013b). The maturing field of ionomics provides not only a set of tools to

understand mineral nutrients but also simultaneous quantification of trace elements (Salt

et al., 2008). Large-scale phenotyping methods allow us to perform high-throughput

analyses of natural variants, mutants and candidate species (Baxter, 2010; Danku et al.,

2013).

4.2 Osmoprotectants can contribute to heavy metals tolerance

Proline is an osmoprotectant often accumulated under osmotic and other abiotic stresses

(Ashraf and Foolad, 2007). For some time the constitutively high proline levels have

been known in a number of halophytic species, as high as 10-20% of shoot dry weight

(Stewart and Lee, 1974; Szabados and Savouré, 2010). Similar to water deficit and

salinity, plants exposed to heavy metal stress accumulate high levels, up to 5-6 times, of

proline (Alia and Saradhi, 1991; Ashraf and Harris, 2004). Many metal tolerant species

such as Armeria maritima, Deschampsia cespitosa, and Silene vulgaris, have

constitutively high levels of proline (Sharma and Dietz, 2006). Proline accumulation

may protect against Hg toxicity by scavenging reactive oxygen species (Wang et al.,

2009). In contrast, loss of proline accumulation increases ROS levels and osmotic

sensitivity (Hong et al., 2000). Studies with Atriplex halimus L. under Cd stress

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demonstrated a two-fold increase in proline levels (Lefèvre et al., 2009), which may

contribute to reduce ROS effects generated by Cd in leaf tissue. Glycinebetaine (GB)

plays a key protective role under water deficit and salinity stress also (Ashraf and Foolad,

2009). A number of halophytes grown on salt-alkalinized soils increase soluble sugars

and betaine under osmotic adjustment (Yang et al., 2012). Osmotic adjustment in

Salicornia europaea and Suaeda maritima is achieved through accumulation of up to five

times the proline and significant increases in glycinebetaine (Moghaieb et al., 2004).

Studies with Atriplex halimus L. demonstrated that Cd increases osmotic adjustment and

induces formation of compatible sugars such as glycinebetaine. Similarly, Armeria

maritima grown on Zn rich soils accumulates proline and betaine (Köhl, 1996).

Free polyamines, specifically spermidine, spermine and diaminopropane, were also found

to increase under Cd induced osmotic adjustment (Lefèvre et al., 2009). The ability to

accumulate soluble carbohydrates to increase salt tolerance in glycophytes has been also

shown, but the role in halophytes is less clear (Gil et al., 2013). In several species, the

application of osmotically active ions such as K to plants grown in the presence of Cd

provided a significant improvement in growth and lowered the toxicity of Cd (Siddiqui et

al., 2012; Shi et al., 2002).

4.3 Sequestration, exclusion and exudation in heavy metals detoxification

Halophytes may sequester high concentrations of salt in the vacuole through maintenance

of high cytosolic K+/Na+ ratios (Glenn et al., 1999; Munns and Tester, 2008). The role of

transporters and the Salt Overly Sensitive (SOS) pathway in A. thaliana has provided

significant advances into understanding salt stress and adaptation (Oh et al., 2010; Ji et

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al., 2013). Loss of the SOS1 sodium/proton antiporter in the halophyte Thellungiella

salsuginea (also referred to as Eutrema salsugineum) conferred sensitivity equal to its

glycophytic relative, A. thaliana (Oh et al., 2009). Another halophytic relative of

Arabidopsis, Schrenkiella parvula (formerly Thellungiella parvula) is adapted to the

hypersaline environment of Lake Tuz in central Anatolia, Turkey (Orsini et al. 2010).

Schrenkiella parvula is capable of growing in hypersaline conditions, in the presence of

sodium concentration six times greater than seawater (Helvaci et al. 2004). Schrenkiella

parvula is capable of surviving concentrations of Na+, K+, Mg2+, Li+, and borate that are

lethal to Arabidopsis thaliana. Recent work has shown that although both share high

level of synteny, S. parvula possess specific genomic structures that likely result in

unique transcriptomes that contribute to its extreme lifestyle (Oh et al., 2014).

Furthermore, these transcriptomes show enrichment for ion-transporters, indicating that

S. parvula relies heavily on exclusion of ions in its toxic environment. Specialized

structures in other halophytes sequester salt in the leaves, isolating toxic levels of heavy

metals from sensitive tissues (Orsini et al., 2010). In the exclusion strategy of

Chenopodium quinoa, young leaves rely on salt bladders where older leaves are almost

entirely dependent on vacuolar sequestration in mesophyll cells (Alatorre et al., 2013). In

spite of the extensive and growing body of knowledge regarding the vacuoles of

halophytes in response to toxic concentrations of NaCl, very little is known on how these

halophytes sequester other toxic ions. Early studies in this area do show promise. A

novel vacuolar H+ ATPase from Tamarix hispida is upregulated in both NaCl and heavy

metal stress and expression studies in yeast showed increased tolerance to both stresses

(Gao et al., 2010). Although specific studies examining vacuolar and cell wall

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sequestration of heavy metals in halophytes are scarce, it can be assumed that some

future studies will demonstrate crosstalk and an interaction between salt and heavy metal

accumulation (Gao et al., 2010).

Some halophytes also possess the ability to exclude toxic ions from tissues with

critical functions through different mechanisms. The presence of oxalates in halophytes

was established decades ago (Karimi and Ungar, 1986; Flowers and Hall, 1978; Yang et

al., 2012). The resistance of some halophytes to heavy metals has also been linked to

oxalate crystals (Lutts et al., 2004). The formation and function of calcium oxalate in

plants has been reviewed by Franceschi and Nakata (2005), while examples of oxalate

based root exudates of Al complexes as well as internal sequestration of Al in vegetative

tissues have been reported by Ma et al. (2001) and Nakata (2003), respectively.

Detoxification of Al through use of Al-tolerant buckwheat that form Al-oxalates in leaf

tissues and produced oxalate root exudates has been proposed as a phytoremediation

strategy, demonstrating the potential of oxalates in the sequestration of toxic ions (Ma et

al., 1997). Heavy metals incorporation into oxalate crystals has also been implicated in

gold, cadmium, copper, lead, and strontium (Lintern et al., 2013; Choi et al., 2001; Yang

et al., 2000; Franceshi and Schueren, 1986).

Trichomes of different species have been found to exclude heavy metals from

metabolically active tissues through formation of Cd and Zn crystals. In tobacco, Cd

treatment increases the trichomes number. Crystals containing calcium and cadmium

were detected in the head cells of trichomes (Choi et al., 2001). Calcium supplementation

in tobacco increased Ca/Zn mineral deposits in Zn exposed plants (Sarret et al., 2006).

Although effective, the mechanism of condensation and excretion remains unclear. It

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should be noted that Cd content of the crystals was calculated to be only 2.3% (Choi et

al., 2001). High concentrations, up to 15-20% of dry weight, have been reported for the

basal compartment of the metal hyper-accumulator Alyssum murale (Broadhurst et al.,

2004). However, the Ni hyper-tolerance of A. murale appears to be specific to Ni and

other heavy metals such as Co are not sequestered in the vacuole (Tappero et al., 2007).

Such findings show that heavy metal tolerance and detoxification mechanisms are often

ion specific.

Within a subset of halophytes a mechanism is present for the secretion of salts

through glandular structures (Thomas et al., 1988; Wagner et al., 2004). Salt glands are a

specific modification of the epidermis for the excretion of salt. The presence of these

glands has only been reported in a few species, but provides an intriguing mechanism for

the exclusion of toxic ions (Flowers et al., 2010). Of the species that do possess salt

glands, up to half of the salt entering the leaf through the transpiration stream can be

excreted (Glenn et al., 1999). Experimental evidence indicates that salt tolerant species

capable of exuding salt also possess the ability to exude other toxic ions, through a

mechanism called “phytoexcretion” (Kadukova et al., 2008; Manousaki and Kalogerakis,

2011). The ability to excrete toxic ions through salt glands has been observed in a number

of salt marsh halophytes (Manousaki and Kalogerakis, 2011). The halophyte Atriplex

halimus L. possesses trichomes that accumulate high levels of Na and Cl ions. When

exposed to 50µM CdCl2 for two weeks, A. halimus exuded more than one third of the

absorbed Cd (Lefèvre et al., 2009). Similarly, the perennial Armeria maritima is high

copper tolerant and is capable of translocating Cu ions through vascular bundles and

excreted by salt glands on the leaf surface (Neumann et al., 1995). A number of species

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within the Spartina genus have been shown to excrete heavy metals in salt crystals

through salt glands (Redondo-Gómez, 2013). S. alterniflora accumulates through leaf

deposition and excretes Cr, Pb, Zn and Hg (Burke et al., 2000; Weis et al., 2002). Metal

excretion has also been reported for other Spartina species, including S. foliosa and S.

anglica (Best et al., 2008; Rozema et al., 1991). Limoniastrum monopetalum is another

halophyte that tolerates high levels of cadmium and lead and excretes these ions through

salt glands as detoxification mechanism (Manousaki et al., 2014). Interestingly, the

domesticated ornamental variety of L. monopetalum accumulated and excreted less Cd

than the wild-type variety, implying a loss of tolerance.

The salt tolerant tree Tamarix aphylla is capable of exuding salt through its leaves

and is resistant to considerable Cd concentrations. Considering that for this species

exudation only accounts for 5% of the total Cd present in the shoots, it is likely that other

mechanisms may concur to its tolerance (Hagemeyer and Waisel, 1998). Studies with

Tamarix smyrnensis Bunge (Kadukova et al., 2008) have also shown very high tolerance

to Cd, up to 30 ppm, and Pb in this species and that uptake, salt gland accumulation and

excretion increase with salinity. It has been demonstrated that plants grown in saline

soils exude 3.4 times more Cd than plants grown in non-saline soils (Manousaki et al.,

2008). These specialized halophytes occur in both xerophytic and salt marsh conditions

and they are a good example of a potential mechanism for avoiding toxic cytosolic levels

of heavy metals. Therefore, phytoexcretion holds great potential for phytoextraction of

some heavy metals from contaminated soils.

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4.4 The role of synthetic chelators in phytoremediation

The addition of synthetic chelators for facilitating the mobility and accumulation of

heavy metals into plant tissues has been well studied. Synthetic chelators such as ETDA,

EDDS and DTPA can increase the accumulation in shoots for metal tolerant species.

EDTA treatments increased Pb accumulation to 218.24 mg kg−1, a 2.69-fold increase,

over controls (Liu et al., 2008). Glycophytic species such as maize and poplar respond

well to soil amended with chelators, by increasing phytoextraction of Pb and mobilization

to leaves (Komárek et al., 2007). Addition of EDTA to contaminated soils increases the

solubility of heavy metals and increases phytoextraction in both metal hyper

accumulators and non-accumulators (Lombi et al., 2001). EDTA-metal complexes

persist for some time, conferring mobility to the chelated heavy metals. Soils amended

with strong and persistent chelators such as EDTA pose risks to the environment,

however (Saifullah et al., 2009). The chelated metals have enhanced mobility and,

therefore, a potential to leach into the groundwater (Meers et al., 2005).

The use of chelators and halophytes in phytoremediation has received limited attention.

When Atriplex nummularia and Zea mays were grown on contaminated soil mixtures

containing heavy metals and two different chelators, EDTA and rhamnolipid, the

halophyte was capable of accumulating more Cu and Pb than the glycophyte. The

halophyte had significantly higher shoot concentrations of Cu, Pb, and Zn when

combined with a chelator in the soil (Jordan et al., 2002). EDTA has also been shown to

increase Cd and Zn accumulation in other species, including macrophytic algae such as

Chara austrailis (Clabeaux et al., 2013) and the halophyte Sesuvium portulacastrum (L.).

S. portulacastrum proves to be an excellent example of a halophyte that functions well

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with synthetic chelators. Experiments using EDTA allowed plants to accumulate up to

3,772 ppm of Pb, while control plants were able to accumulate only 1,390 ppm (Zaier et

al., 2014). Management practices that foster beneficial soil microbes can also

increase ion availability to plants and enhance phytoextraction (Marchiol and Fellet,

2011). Trichoderma and humic substances contained in compost can increase the

phytoextraction ability of plants to remove ions such as Cd from contaminated soils

(Fiorentino et al., 2013).

4.5 Halophytes and hydrocarbons

Phytoremediation does not refer solely to heavy metals and ions and an increasing

number of investigations have focussed on plants and hydrocarbons also. Polycyclic

aromatic hydrocarbons (PAHs) are anthropogenic sources of pollutants that can

contaminate atmospheric, marine and soil environments. The harmful effects of oil spills

on flora, fauna and humans are examples of significant environmental impacts as well as

consequences for human health that hydrocarbons may have (Aguilera et al., 2010).

PAHs can accumulate in coastal soils (Tolosa et al., 2004) and in plants grown with

wastewater or on contaminated soil (Tao et al., 2006). E-waste recycling in developing

countries can be a particularly acute source of PAHs. Vegetables grown on soils enriched

of these pollutants have resulted to be unsafe for consumption (Luo et al., 2011; Wang et

al, 2012). Spartina densiflora, a grass common to salt marshes, has a high tolerance to

the PAH phenanthrene and a high rate of extraction from the soil (Redondo-Gómez et al.,

2011a). Similarly, the halophyte Salicornia fragilis has been shown to accumulate PAHs

in shoot tissues after exposure to contaminated sediments (Meudec et al., 2006; Meudec

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et al., 2007). The salt marsh halophyte Halimione portulacoides accumulates Zn, Pd and

Cu and several studies indicated that enrichment of PAH in conjunction with Cu

facilitated increased Cu uptake (Cacador al., 2000; Almeida et al., 2008). Considering

the ability of edible species to bioaccumulate PAHs, halophytes present a possible

alternative for remediating contaminated soils without consequences/risk for human

health.

5. Unveiling possible conflicts in tolerance mechanisms to NaCl and heavy metals

Broader utilization of halophytic species in soils that are degraded by both high salt and

heavy metals is limited by our rather modest understanding of the fundamental

mechanisms governing the tolerance to these two environmental stressors, including their

mode of interaction. If we are to broaden the use of these species to marginal

environments that cannot be used for standard agricultural purposes it will require a

deeper understanding of the mechanisms of tolerance and the ion-specific and specie-

specific differences. Responses to heavy metals and salt vary between ion and species

greatly. This complexity presents a challenge in understanding the mechanisms at play

but also implies the existence of numerous genetic resources for engineering tolerance.

Kosteletzkya virginica for example is a halophyte from the Malvaceae family with

potential for biofuels considering its high yield in tidal marshes, reaching up to 1500

kg/ha, with a high protein and seed oil content up to 20% (Ruan et al., 2008). The

response of Kosteletzkya virginica to various heavy metals varies with metal and

concentration of salt. Specifically, Cd and Zn have opposite effects on cytosolic K level,

which is essential to osmotically balance sodium ions sequestered in the vacuole. It was

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found that cadmium reduced the uptake of Na under salt treatment and increased K

concentration in leaves. In contrast, Zn increased Na translocation and reduced leaf K

levels (Han et al., 2012). By increasing NaCl, Cd accumulation was reduced but still

unable to alleviate Cd-induced growth effects. The presence of NaCl in conjunction with

Cd was shown to reduce senescence, decrease oxidative stress and reduce ABA levels

(Han et al., 2013), indicating that for some halophytes, the presence of NaCl may be

required to cope with oxidative stress such as those induced by heavy metals. Similarly,

significant levels of Cu, Cd, and Pb were found to accumulate in stems and leaves of

Halimione portulacoides, a small evergreen shrub found in coastal salt marshes

(Reboreda and Cacador, 2007). Roots of H. portulacoides accumulated significant

amounts of Zn, Cu, Ni, and Co and correlated well with soil salt concentrations (Milić et

al., 2012).

Competition between heavy metals, Na+ and Cl- ions has been reported for the salt

marsh grass Spartina alterniflora, which is also known for its cold tolerance. High

phytoextraction rates have been reported for S. alterniflora in above ground tissues for

both Cr and Fe (Windham et al., 2003; Nalla et al., 2012). However, as salinity

increased, S. alterniflora decreases the amount of Zn and Pb in excreted salts, showing a

reduction in uptake of these heavy metals and possibly competition with Na+ and Cl- ions

(Mahon and Carman, 2008).

In the absence of NaCl, the xerohalophyte Salsola kali L. (synonym Kali turgida)

can accumulate Cd2+ at high levels in the roots (400 µg Cd2+ g−1 DW), while

accumulating much lower concentrations in the shoots (85 µg Cd2+ g−1 DW). In contrast,

in the presence of NaCl root Cd2+ was decreased, but shoot levels remained the same.

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With the addition of EDTA, shoots accumulated twice as much Cd2+ (Ben Rejeb et al.,

2013). While the physiological and molecular reasons underlying these responses are

currently unknown, these findings indicate that S. kali is a prime candidate for

phytoextraction of Cd2+ and highlight a potential role for EDTA in enhancing stable

uptake of contaminants.

Atriplex is a genus belonging to the Chenopodioideae subfamily in the

Amaranthaceae family and includes a number of halophytic species tolerant to heavy

metals. This genus contains species adapted to both xerophytic and seashore

environments. Atriplex halimus L., commonly known as Mediterranean Saltbush, grown

with Cd and Pd demonstrated significant tolerance (Lutts et al., 2004). Increasing

salinity resulted in increased cadmium uptake where lead uptake did not seem to be

affected (Manousaki and Kalogerakis, 2009). Opposite effects have been observed in

different environments. Chlorine salinity (NaCl and KCl) reduced Cd accumulation in A.

halimus while addition of NaNO3 increased Cd accumulation in leaves (Lefèvre et al.,

2009). A. halimus shows improved growth in soils contaminated with Cd when grown in

conjunction with NaCl or KCl. NaCl alleviated the phytotoxicity caused by Cd stress

likely through enhanced osmoprotectants antioxidative activities (Chai et al., 2013).

Different responses within the same family could be attributed to different

lifestyles also. The Aizoaceae family is mostly composed of species from arid and semi-

arid environments and often have succulent leaves. Two halophytes from this family,

Sesuvium portulacastrum and Mesembryanthemum crystallinum, have been challenged

with cadmium and have been shown to be tolerant to nickel (Amari et al. 2014). Both

species were able to accumulate large amounts of Cd and had higher concentrations of Cd

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in the roots than in the shoots, a common trait of metal tolerant species. S.

portulacastrum was significantly more tolerant than M. crystallinum (Ghnaya et al 2005),

however. While both species are members of the Aizoaceae and halophytes, their ability

to respond to toxic levels of Cd differed. This may be due in part to their life cycle where

S. portulacastrum is a perennial and M. crystallinum is an annual species. Additional

adaptation mechanisms may differentiate the two species, also. M. crystallinum has an

uncommon metabolism that allows the plant to switch from C3 metabolism to CAM

under high water or salt stress (Tallman et al., 1996) and this may confer greater

sensitivity to heavy metals. In contrast, M. crystallinum utilizes its distinct mechanisms to

tolerate and accumulate high levels of Cu and Zn (Thomas et al., 1998; Kholodova et al.,

2005). Indeed, a number of genes involved in the CAM metabolism were both inducible

by NaCl and Cu at various stages of development, indicating some overlap in tolerance

mechanisms (Thomas et al., 2004). It was later found that Cd had a limiting effect on

potassium and calcium uptake and the growth inhibition can be compensated by

increasing calcium availability (Ghnaya et al., 2007).

6. Specificities of some halophytes

In recent years significant progress in understanding the links between NaCl and heavy

metal responses has been made with species in the Poaceae family. The genus Spartina

includes C4 perennial grasses often found in coastal area or inland salt marshes.

Redondo-Gómez, (2013) reviewed a number of these species and their ability to tolerate

and accumulate heavy metals. Spartina argentinensis, S. densiflora, S. maritima all have

been shown to accumulate heavy metals. In particular, S. densiflora and S. maritima

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show potential in phytoremediation for their pronounced capacity for phytostabilization

and accumulation of As, Cu, Fe, Mn, Pb, and Zn (Cambrollé et al., 2008). It has been

reported that Zostera noltii (Hornem) and S. maritima (Curtis), are sources of heavy

metals in unpolluted estuaries (Couto et al., 2013). These halophytes appear to have the

ability to concentrate and localize heavy metals, even when their concentrations are low

in the surrounding sediments. Salt marshes are particularly susceptible to heavy metal

contamination and S. maritima has anti-oxidant feedback responses in the presence of

heavy metals. For this specific function, S. maritima has been proposed to be used as a

biomarker species through monitoring of its anti-oxidant feedback (Duarte et al., 2013).

Other Spartina species have been reported for their exceptional accumulation ability. S.

argentinensis has been shown to accumulate over 1.0 mg g-1 of Cr, demonstrating a

strong hyperaccumulator phenotype (Redondo-Gómez et al., 2011b).

Atriplex species have also been assessed for their ability to hyperaccumulate Ni,

Cu, Pb, and Zn. A. hortensis var. purprea, var. rubra, and A. rosea did not hyper-

accumulate metals in vegetative tissues, but were able to bioconcentrate them in root

tissues (Kachout et al., 2012). Field experiments with A. halimus in semi-arid conditions

showed a capacity for phytostabilization on heavy metal contaminated soils, through

concentration of ions in vegetative tissues (Clemente et al., 2012).

Remarkable tolerance to heavy metals has been reported for the genus Halimione

(a member of the Chenopodioideae subfamily in the Amaranthaceae family). H.

portulacoides tolerates high levels of Zn, remaining unaffected at concentrations as high

as 1500 mg Zn kg-1 (Cambrollé et al., 2012a). The mechanisms that mediate Zn

tolerance in this species do not cross over into Cu tolerance, however. While H.

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portulacoides could tolerate 15 mmol Cu L-1 in soils, it showed significant reduction in

biomass at higher concentrations (Cambrollé et al., 2012b). The ability of H.

portulacoides to accumulate high concentrations of Zn makes this species an excellent

candidate to recover Zn contaminated soils.

S. portulacastrum seedlings were shown to tolerate up to 1mM Pb2+ and to

accumulate it in the shoots, demonstrating a robust ability to phytoextract Pb (Zaier et al.,

2010a,b). Field studies have also shown the great potential of a halophytic member of

the Aizoaceae family, Sedum plumbizincicola, which in successive cropping over two

years was able to significantly reduce levels of Zn and Cd in tested soils, resulting in

increased microbial biomass (Jiang et al., 2010). Additional traits to be considered for

these species are those concerning their vegetative habit. The salt marsh plant

Sarcocornia perennis may be suitable for phytoremediation of heavy metals because of

its low root mass turnover and ability to phytostabilize (Duarte et al., 2010).

7. Concluding remarks and a look forward

Although a number of plants are currently being considered for use in the phytoextraction

of heavy metals from contaminated soils (Table 1), several unsolved issues remain. For

phytoextraction to be a viable solution, plants need to possess the ability to uptake

specific ions and accumulate them at high concentrations while not suffering deleterious

effects due to their toxicity. Moreover, as the goal of phytoextraction is to remove a

contaminant from the environment, hyperaccumulators must be harvested and disposed

properly. The disposal aspect of this process poses a number of problems. Different

methods are available for this purpose: incineration, direct disposal, ashing and liquid

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extraction. At this time incineration is the mostly widely accepted method because of the

feasibility of the procedure and economics (Sas-Nowosielska et al., 2004), also if pyro-

gasification is considered to have lower environmental impact thanks to metal

immobilization in the solid phase (Vervaeke et al., 2006; Lievens et al., 2008). Disposal

of combustion toxic by-products can add significant costs to the processing of ash wastes

after incineration. Recent studies have demonstrated the potential for this ash waste for

use as fertilizer if diluted to acceptable levels (Bonanno et al., 2013) or for metal

recovery by using hydrometallurgical routes, such as the carried-in-pulp method

proposed by Alorro et al. (2008).

In principle, halophytes have a lifestyle that seems to overlap with mechanisms

that confer heavy metal tolerance. Their adaptive mechanisms of exclusion,

sequestration, exudation, and metabolic adjustment provide a robust system to survive in

toxic and extreme environments in general. A number of promising candidates exist that

do possess lifestyle adaptations that confer tolerance beyond sodium and chloride. It has

been proposed that a pragmatic approach should be used to engineer crops to be

hyperaccumulators (Wu et al., 2010). Engineered hyperaccumulator crops would still be

limited to non-saline soils and would tend to perform poorly in stressful environments,

however. The same techniques that have been proposed to develop hyperaccumulating

crops could apply, possibly more effectively, to halophytes.

In a broader context, utilization of marginal soils and remediation of productive

lands rendered unsuitable by/for agriculture through use of halophytic species could

represent an important avenue to pursuit to avoid conflicts between requirements for

more food, food quality, biofuels production, land abandonment and consequent

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desertification. In this respect, a very clear case has been made for an ethical and

sustainable production of biofuels in such a way that it does not foster competition

between existing food crops (Bressan et al., 2011). Extremophile species present a

solution to this dilemma. Moreover, as recently addressed (Sonnino et al., 2014), a

sustainable food security framework cannot rely anymore on individual/independent

component of the food system and must look at innovative solutions that

better/holistically integrate economic, social and ecological dimensions of the system. A

functional component of this system referred to the use halophytes is presented in Figure

1. This figure represents a proposed action plan for implementing metal tolerant

halophytes as agronomic tools to be used on contaminated soils. In recent years,

genomes and biotechnology tools have progressed rapidly, vastly increasing the potential

applications of halophytes in many unexplored contexts (Dassanayake et al., 2011; Oh et

al., 2012; Bressan et al., 2013; Batelli et al., 2014). The lack of a clear understanding of

the relationship between salt tolerance and metal tolerance in these species, and their

potential application in diverse environments, emphasizes the need for further studies in

this area.

ACKNOWLEDGEMTS

This work was supported by Italian “Ministero dell'Istruzione, dell'Università e della Ricerca” through the PON Research and Competivity 2007-2013 (ENERBIOCHEM- PON01_01966).

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Zaier H, Ghnaya T, Lakhdar A, Baioui R, Ghabriche R, Mnasri M, Sghair S, Lutts S, Abdelly C. 2010. Comparative study of Pb-phytoextraction potential in Sesuvium portulacastrum and Brassica juncea: Tolerance and accumulation. Journal of Hazardous Materials 183: 609–615.

Zaier H, Ghnaya T, Ghabriche R, Chmingui W, Lakhdar A, Lutts S, Abdelly C. 2014. EDTA-enhanced phytoremediation of lead-contaminated soil by the halophyte Sesuvium portulacastrum. Environmental Science and Pollution Research 21: 7607–7615.

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Zaier H, Mudarra A, Kutscher D, de la Campa MRF, Abdelly C, Sanz-Medel A. 2010. Induced lead binding phytochelatins in Brassica juncea and Sesuvium portulacastrum investigated by orthogonal chromatography inductively coupled plasma-mass spectrometry and matrix assisted laser desorption ionization-time of flight-mass spectrometry. Analytica Chimica Acta 671: 48–54.

ACCEPTED MANUSCRIP

T

Ta

ble

1:

Sp

eci

es

Dis

cuss

ed

: S

um

ma

ry o

f sp

eci

es

dis

cuss

ed

in t

he

re

vie

w,

inte

ract

ion

wit

h h

ea

vy

me

tals

or

con

tam

ina

nts

, an

d r

ele

va

nt

refe

ren

ce.

Po

ten

tia

l so

urc

es

or

con

cen

tra

tors

are

no

ted

wit

h a

n “

S”

wh

ile

to

lera

nt

spe

cie

s a

re n

ote

d w

ith

a “

T”,

acc

um

ula

tin

g a

nd

hyp

era

ccu

mu

lati

ng

spe

cie

s a

re n

ote

d w

ith

an

“A

”, a

nd

sp

eci

es

use

d f

or

rem

ed

iati

on

of

sali

ne

so

ils

are

no

ted

wit

h a

n “

N”.

Spe

cies

Nam

e R

esp

onse

Hea

vy M

etal

s R

efer

ence

Arm

eria

mar

itim

a T

/A

Cu,

Zn

(K

öhl

, 1

996

; Neu

man

n e

t al

., 1

995

).

Atr

iple

x ha

limus

T

/A

Cd,

Pb,

and

Zn

(Lu

tts e

t al

., 2

004

; Cle

men

te e

t al

., 2

012

) A

trip

lex

len

tifo

rmis

N

N

a (G

len

n et

al.,

200

9)

Atr

iple

x nu

mm

ula

ria

T/A

C

u, P

b, a

nd Z

n (J

orda

n et

al.,

20

02).

Ch

eno

pod

ium

qu

inoa

N

N

a (A

lato

rre

et

al.,

201

3).

H

alim

ion

e p

ortu

laco

ides

T

/A

Co,

Cu

, N

i, an

d

Zn

(M

ilić

et

al.,

201

2)

Ko

stele

tzky

a v

irg

inic

a

T/A

Z

n

(Han

et a

l., 2

012

).

Lim

oni

ast

rum

mo

nop

eta

lum

T/A

C

d, P

b (M

ano

usak

i et

al.,

201

4).

M

ese

mbr

yant

hem

um

cry

sta

llin

um

T/A

C

d, C

u,

Zn

(T

hom

as et

al.,

199

8; K

ho

lodo

va et a

l., 2

005

; Gh

nay

a et

al

200

5)

Sal

icor

nia

bra

chia

ta

T/A

A

s, C

d,

and

Ni

(Sh

arm

a et a

l., 2

009

) S

alic

orn

ia fra

gili

s T

P

AH

(M

eud

ec e

t al

., 2

007

) S

alic

orn

ia m

aritim

a T

C

u an

d Z

n (M

ilić

et

al.,

201

2).

Sal

sola

ka

li

T/A

C

d (d

e la

Ro

sa et a

l., 2

004

) S

arco

corn

ia p

ere

nn

is T

C

u, P

b (D

uar

te et

al.,

20

10).

S

edu

m p

lum

biz

inci

cola

T

/A

Cd,

Zn

(J

iang

et a

l., 2

010

) S

esu

viu

m p

ort

ula

cast

rum

T/A

/N

Cd,

Na,

Pb

(Z

aier

et

al.,

201

0a,

b; R

abhi

et a

l., 2

01

0 G

hnay

a e

t al 2

00

5)

Spa

rtin

a f

olio

sa

T/S

H

g (B

est e

t al

., 20

08)

S

part

ina

alte

rnifl

ora

T

/A

Cr,

Hg,

Fe,

Pb,

Z

n a

nd P

AH

(B

urk

e et

al.,

200

0; W

eis e

t al

., 20

02;

Red

on

do-G

óm

ez e

t al

., 20

11a

)

ACCEPTED MANUSCRIP

T

Spa

rtin

a a

nglic

a

T/A

F

e, M

n a

nd

Zn

(R

oze

ma

et a

l., 1

991

) S

part

ina

arg

en

tinen

sis

T/A

C

r (R

edo

ndo

-Gó

mez

et a

l., 2

011

b)

Spa

rtin

a d

ens

iflo

ra

T/A

A

s, C

u,

Fe,

Mn

, P

b, a

nd

Zn

(C

ambr

ollé

et a

l., 2

008

)

Sua

ed

a m

ariti

ma

T

/A/N

A

s, C

d,

Cu

, Fe,

M

n, N

a, P

b, a

nd

Zn

(Rav

ind

ran

et a

l., 2

007

; K

öhl,

199

6; C

amb

rollé

et

al.,

200

8)

Sul

la c

arn

osa

N

N

a (J

lass

i et

al.,

201

3)

Tam

arix

ap

hylla

T

/A

Cd

(Hag

emey

er a

nd

Wai

sel,

199

8).

Tam

arix

smyr

nens

is

T/A

C

d an

d P

b (M

ano

usak

i et

al.,

200

8; K

aduk

ova

et

al.,

200

8)

Zos

tera

no

ltii

T/S

C

o, C

u,

Pb

an

d Z

n

(Co

uto

et a

l., 2

013

).

ACCEPTED MANUSCRIP

T

Fig. 1. Schematic representation of potential applications and indirect benefits arising

from utilization of halophytes on contaminated soils.

ACCEPTED MANUSCRIP

T

ACCEPTED MANUSCRIP

T