Plants response to high soil Zn availability.

Feasibility of biotechnological improvement

1 2 3
Silvana Irene Torri , Marisol Natalia Cabrera , Martin Torres Duggan
1
Facultad de Agronoma, Universidad de Buenos. Aires, Av. San Martn 4453, Ciudad Autnoma de Buenos Aires, C1417DSE Argentina
2
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Giraldes 2160, Ciudad Autnoma de Buenos Aires,
C1428EGA Argentina
3 Tecnoagro, Girardot 133, Ciudad Autnoma de Buenos Aires, C1427AKC Argentina.
torri@agro.uba.ar

Abstract

Zinc (Zn) is a trace element required in small amounts for most biological systems, although it can be toxic when absorbed
in excess. Some plants have evolved the ability to tightly regulate their internal Zn concentrations in spite of high soil
availability, a process called zinc homeostasis. A portion of these species can even hyperaccumulate Zn, with a potential
use to restore contaminated soil environments. A range of gene families that are likely to be involved in Zn transport have
been identified. The growing application of genetic technologies may led to increase plant Zn resistance strategies, as well
as increased uptake of this element from polluted environments. These characteristics may be used for phytoremediation
techniques. This Chapter provides a broad overview of different plant strategies to deal with stress originated from high
Zn availability in soils. Current status of biotechnological improvement and its future prospects are reviewed.

1. Introduction

Zinc (Zn) is a trace element required in small amounts for most biological systems, including plants and animals. The term
trace element is widely used for those elements that occur at relatively low concentrations in the dry matter of living
1
organisms, usually under 100 mg kg . However, this term is imprecise because it is only based on the concentration of the
element in the plant or animal system, regardless its role.

Several trace elements have been reported to be necessary to complete the vegetative or reproductive stage of plants life
cycle, cannot be replaced by other elements, or are directly involved in plant metabolism (Arnon, Stout 1939). These trace
elements are termed micronutrients, and include boron (B), chlorine (Cl), copper (Cu), iron (Fe), manganese (Mn),
molybdenum (Mo), nickel (Ni) and zinc (Zn). Other trace elements such as arsenic (As), cadmium (Cd), mercury (Hg) and
lead (Pb) have no known biological role in plants but can enter plant cell through existing mineral uptake pathways. These
elements are non-essential, and may form unspecific complex compounds in plant cell causing damage to normal
physiological processes (Sytar et al, 2012).

The importance of Zn for plant cell growth and development has been widely reported for the last century. The fact that
Zn does not take part in oxido-reduction reactions, combined with its geometry makes it a suitable cofactor for many
enzymes. Zinc is the only trace element involved in all six classes of enzymes: oxido-reductases, transferases, hydrolases,
lyases, isomerases and ligases (Barak, Helmke 1993). In plants, enzymes either containing or activated by Zn are involved
in a number of physiological processes and cellular metabolism, including protein synthesis; enzyme activation;
metabolism of auxins, carbohydrates, lipids and nucleic acids and antioxidative defense (Marschner 1995; Broadley et al.,
2007). Zinc is also required to regulate plant tolerance of environmental stress, such as high light intensity, high or low
temperatures (Cakmak 2000). The essentiality of Zn in plants was first reported for maize, and afterwards for barley and
dwarf sunflower (Maz, 1915; Sommer, Lipman 1926).

Zinc is probably one of the most ubiquitous elements in nature. Total Zn concentration in pristine soils is largely
dependent on the parent material and the chemical and physical weathering processes in which the soil has developed
(Mason, Moore 1982). In non-contaminated agricultural soils, total Zn concentration usually ranges from 50 to 300 mg
1
kg (Adriano, 2001).

Anthropogenic release of Zn to the environment has greatly increased soil Zn concentration of urban and agricultural soils.
Human activities such as industry, mining, metalliferous smelting processes, atmospheric deposition, together with
improper agriculture practices such as overuse of chemical fertilizers and pesticides, industrial or sewage sludge
amendments, or wastewater irrigation have considerably contributed to increase total Zn concentration in soils (Adriano
2001; Garbisu, Alkorta 2003). Friedland (1990) estimated that the ratio of Zn emissions arising from anthropogenic and
natural inputs were greater than 20 : 1. In certain polluted areas, soil Zn concentration has been reported to be more than
1
1000 mg Zn kg dry soil (Castro-Larragoitia et al, 1997). Zn contaminated soils cause injury to soil microorganisms and
reduce crop yield (Hassan and Aarts, 2011). Moreover, high Zn concentration in soils may cause Zn accumulation in the
edible parts of crop plants, representing the principal route of entry into the human food-chain. Unlike organic pollutants,
trace elements cannot biodegrade as a result of biological or chemical processes and, consequently, persist in the
environment for longer periods of time than in other compartments of the biosphere (Lasat, 2002). Therefore, trace
elements are a group of soil pollutants of great concern.

2. Bioavailability of Zn in soils

The evaluation of total Zn concentration in soils is a useful index of the degree of Zn pollution. The evaluation of
background data on soils unaffected by human activities can be useful to determine the rate of soil pollution. Worldwide,
total soil concentration ranges and regulatory guidelines for some trace elements have been proposed (NJDEP 1996; Riley
et al. 1992). However, total Zn concentration provides little or no information on the fraction that plants can absorb (Wolt,
1994). It is now widely recognized that Zn bioavailability strongly depends on its specific chemical forms and on its binding
state in the soil. Many researchers have reported that total Zn concentration in soils does not correlate with the Zn
bioavailable pool (McBride et al., 1997; Torri, Lavado 2009).

When Zn reaches the soil surface, it may distribute between different soil fractions (Marschner 1995, Torri et al 2012). The
distribution of Zn is determined by soil-specific precipitation, complexation or adsorption/desorption reactions. In soils, Zn
2+ +
can be found as free ions [Zn , Zn (OH) ] or in soluble organic complexes in the soil solution, associated with clay particles,
humic compounds or Fe and Mn hydroxides, precipitated with secondary minerals, or within the matrix of soil primary
minerals (Shuman 1991). Bioavailable Zn fractions are usually considered to be soluble and exchangeable Zn and Zn-bound
to organic complexes, although some plants may develop mechanisms by which Zn is selectively absorbed (Torri, Lavado
2009).

Soil chemical properties or changes of environmental conditions may also determine Zn distribution among soil
constituents and, therefore, Zn availability to plants. Soil pH is considered to be the main soil factors controlling the
amounts of plant-available Zn in soils. At high soil pH, Zn is readily adsorbed onto cation exchange sites. Conversely, Zn
solubility increases under acidic conditions (Kabata-Pendias 2001). In accordance with these changes in Zn availability,
plant uptake of Zn increases as soil pH decreases. Other soil characteristics that affect Zn bioavailability include soil
organic matter content, clay content, amorphous iron or manganese oxides, calcium carbonate concentration, redox
conditions, microbial activity in the rhizosphere, soil moisture, climate and concentration of macronutrients or other trace
elements (Alloway 2009). Soil texture was also reported to affect the levels of Zn in the soil solution (Meers et al, 2006).

Nonetheless, the distribution of Zn among soil fractions is subjected to a dynamic equilibrium (Norvell, 1991). For
instance, plant roots directly remove Zn from the soil solution, which is rapidly replenished from Zn adsorbed in easily
desorbed forms. Plant roots may also contribute to increase Zn availability through changes in rhizosphere pH or by root
exudation of organic acids or phytosiderophores (Reichman, Parker 2005). In soils with high organic matter content,
soluble Zn was reported to increase at soil pH 77.5, as a result of an increase in the concentration of chelating agents in
the soil solution (Wong et al, 2007). Inorganic precipitates were reported to reduce Zn availability in soils at high pH (Torri,
Lavado 2008).

Several experiments have shown that Zn bioavailability and phytotoxicity depends on the concentration of free Zn species
in the soil solution (Weng et al., 2001). These results were probably due to the fact that free Zn is one of the most
frequent phytotoxic elements after free aluminum and free manganese (Chaney, 1993). Apart from the free divalent
cation, the soil solution may contain Zn as inorganic or organic complexes (Holm et al., 1995). Nevertheless, the
10 6
concentration of soluble forms of Zn in soils is low, usually between 4 10 and 4 10 M (Barber, 1995), even in Zn-
polluted soils (Wu et al. 2000). Free Zn typically accounts for up to 50% of the soluble Zn fraction. Although in many
studies considerable amounts of soluble or exchangeable Zn were anthropogenically added to soils (through waste-water
irrigation, biosolids disposal), with the passage of time Zn transforms gradually from more active and available fractions
into less available species (Ma and Uren 2006). In these soils, the largest fraction of Zn was reported to occurr primarily as
inorganic precipitates, whereas the concentration of water-soluble and exchangeable Zn was usually very low (Schalscha
et al. 1999; Torri, Lavado 2008), a phenomenon known as aging. Therefore, the observed Zn phytotoxicity in Zn-polluted
2+
soils that exhibit low concentration of water-soluble and exchangeable Zn is soil high buffer capacity: as free Zn is taken
up by the plant more Zn solubilizes to the soil solution through different chemical reactions to maintain the initial Zn
concentration. In this case, the total solution Zn concentration may not change appreciably whilst Zn is taken up by plants.

3. Zn plant uptake

Plant uptake of essential trace elements, such as Zn, is a complex process that is regulated by metabolic mechanisms at
2+
the soil-root interface. As discussed in a previous section, Zn is generally taken up as a free divalent cation (Zn ) or as
 +
monovalent ZnOH at high soil pH (Marschner 1995). Some studies indicated that uptake of Zn by barley and lettuce not
only depended on the free-Zn concentration in the soil solution, but was also influenced by the level of soluble Zn-
complexes (Bell et al. 1991, McLaughlin et al. 1997).

Two hypotheses have been postulated to explain the contribution of aqueous organic complexes to Zn uptake by plants: i)
uptake is limited by the diffusion rate of free Zn to the root surface, so the presence of Zn-labile organic complexes
increase the diffusive supply of Zn to roots, and free Zn is uptaken as a result of complex dissociation in the diffusive
boundary layer of soil-root interface (Degryse et al, 2006) or ii). Zn-complexes are directly absorbed by plant roots (Wang
et al., 2009). Zn- phytosiderophores complexes have been demonstrated to be absorbed by maize (von Wren et al. 1996).

Although Zn is an essential trace element for plant growth, high Zn concentration in plant tissue may originate toxicity
symptoms. However, plant stress originated by high Zn availability in soils is far less widespread than stress produced by
Zn deficiency. For normal growth and development, plants must keep the concentration and speciation of intracellular Zn
of essential micronutrients like Zn within an optimal range. This physiological mechanism is known as metal homeostasis,
helping plants to survive both at metal deficiency or at higher metal concentrations. Only when this control mechanism
has reached its capacity, Zn cell concentration rises above certain threshold level and toxicity effects appear (Sinclair,
Kramer 2012). The idea of a threshold toxicity level is often used to establish the point at which trace elements cause
significant growth decrease. Threshold values are often defined as element concentration corresponding to a yield
decrease of 10% (Alloway 1995). In most crops, typical leaf Zn concentration required for adequate growth or maximal
1
yield is in the range 15 -30 mg Zn kg DW (Marschner, 1995). Toxicity thresholds are highly variable within species:
1
although toxicity symptoms usually appear when leaf Zn concentrations exceeds 300 mg Zn kg leaf DW, some crops may
1
exhibit toxicity symptoms when leaf Zn concentrations is below 100 mg Zn kg DW (Marschner, 1995). Therefore, plant Zn
concentration should be maintained in various tissues and compartments within a relatively narrow range to avoid
deficiency or toxicity effects.

4. Plant response to high soil Zn availability

The presence of high trace elements availability in soils exerts a metal-induced stress on plant populations. Stress is
generally defined as the reaction of a biological system, in this case plants, to extreme environmental factors that,
depending on their intensity and duration, may cause significant changes in the system (Nilsen, Orcutt 1996). Excessive Zn
accumulation in root or shoot cells of sensitive plants may induce oxidative stress by the inactivation of proteins by
adventitious binding of Zn (Sarret et al. 2006). The latter may interfere with important metabolic processes, like
photosynthesis (Chaney 1993). Depending on the severity of Zn exposure, stress may induce physiological disorders
resulting in reduced biomass, leaf chlorosis, inhibited root growth, and physiological disorders, often leading to plant
death at excessive exposure. Other plant species, due to natural selection, are able to colonize polluted areas, usually
forming a specific local flora adapted to growth and reproduction in adverse conditions. As a result of ecophysiological
adaptation, some plant species have developed strategies to resist high Zn stress exposure which can be grouped into one
of three categories: i) excluder strategy, ii) tolerant strategy and iii) hypertolerant strategy. These strategies are metal-
dependent and plant species-dependent, and can operate until biochemical resistance of plant cells exist. Nonetheless,
whichever strategy plants use to limit the negative effects of Zn toxicity, a complex network of Zn transport, chelation, and
intracellular sequestration processes have to operate to maintain Zn homeostasis in plant cells.

4.1 the excluder strategy

Plants that developed this strategy try to keep low Zn concentrations in the roots across a wide range of soil Zn
concentrationl (Baker, 1981). This is achieved by either restricting Zn soil bioavailability or by reducing Zn uptake.

Zinc can enter the plant symplast by diffusion, by passive transport through channel proteins, or by active transport
through carrier proteins, termed metal transporters (Eide, 2006; Claus, Chavarria-Krauser. 2012) . Zn is most likely crossing
the plasma membrane via members of the ZIP transporter family (Zinc-regulated transporter, Iron-regulated transporter
Protein). Members of the ZIP gene family are capable of transporting a variety of cations, including cadmium, iron and
manganese (Guerinot, 2000). Under high Zn availiability soil conditions, plants will try to inhibit excessive Zn influx through
down regulation of Zn transporters

Another way of avoiding the entry of Zn into the plant is by binding Zn to specific root exudates. Stress stimulates roots to
exude an array of compounds, including soluble and non-soluble organic compounds (Bais et al, 2006). Root exudates can
form Zn complexes in the rhizosphere, making them unavailable to the plant or lessening Zn toxicity.

Most plants have mycorrhizal fungi associated, with an increased capacity to absorb water and nutrients from the soil.
Mycorrhizal symbiosis is affected by anthropogenic stressors including metals (Entry et al. 2002). The presence of
arbuscular mycorrhiza in plant roots may also provide a Zn excluder barrier, immobilizing Zn in vacuoles (Weissenhorn et
al., 1995) or preventing its transport to host plants through the formation of organic complexes (Leyval et al., 1997). Plants
may also modify the composition of the root cell wall to prevent Zn from entering the plant, enhancing its Zn-binding
capacity (Neutelings, 2011).

Remarkably, mMechanisms involved in the exclusion processes are much weaker than those developed by roots for Zn
absorption under deficient conditions. So, high Zn availability in soils is a stronger stress to plants than Zn deficiency. In
highly Zn- polluted soils, it is often difficult to exclude Zn from plant roots due to the high Zn soil availability. When the
tolerance limits of excluder species are exceeded, phytotoxic effects appear, resulting in plant death.

4.2 the tolerant strategy

Some plant species have evolved different mechanisms to tolerate high Zn availability in soils. Even within one plant
species, more than one mechanism could be operating.

When exposed to excess Zn, some plant species accumulate Zn in their tissues, reducing the damage by storing Zn
in unavailable sinks. Plants may accumulate moderate to high amounts of Zn in roots, or Zn may be translocated from
roots to above ground tissues via xylem. In either type, Zn that gets past the plasma membrane is detoxified by physical
sinks, like chelation in the cytosol, compartmentation in vacuoles, or confinement in the apoplast in order to reduce an
undesired interaction with cellular compounds.

Of all mechanism involved in preventing or lessening the toxicity effects of Zn, compartmentation in vacuoles is regularly
reported as the most probable. Zinc sequestration in the vacuoles of root cells may limit the translocation from root to
shoot (Lasat et al. 1998) whereas xylem unloading in the shoots and Zn sequestration in the vacuoles of leaf cells may
contribute to translocation to above ground tissues (Palmgren et al. 2008).

4.3 the hypertolerant strategy.

Some plant species growing on naturally or anthropogenically trace elements contaminated soils were found to be
extremely tolerant to exposure to high soil availability of trace elements. In these plants, the concentration of some trace
elements accumulated in the aboveground biomass was found to be up to four orders of magnitude higher than in other
plants. These naturally occurring plant species have evolved the ability to take up, tolerate and accumulate trace elements
to exceptionally high concentrations in their aerial tissues and maintain metal homeostasis (Reeves, Baker 2000).
1
The term hyperaccumulator was used to describe plants that accumulate more than 1000gNig of dry weight in their
shoots growing in its natural habitat (Brooks et al., 1977). The phrase growing in its natural habitat means that
hyperaccumulators must achieve their high metal concentrations while remaining healthy enough to maintain a self-
sustaining population. Subsequently, the term hyperaccumulator has expanded to other trace elements such as Cd, Co, Cr,
Cu, Mn, Pb, Se and Zn, and more than 500 plant taxa have been reported to be hyperaccumulators as reviewed by Reeves
and Baker (2000). Plant trace element hypertolerance and/or hyperaccumulation are clear ecophysiological adaptations to
metalliferous soils.

The first physiological step in the accumulation process is an enhanced Zn uptake into the root cells. Zinc may enter the
plants through the plasma membrane of the root endodermal cells, through the symplast, or by crossing the root apoplast
through intercellular spaces. A reduced vacuolar sequestration in root cells is the next step, together with an enhanced
symplastic transport through the root towards the vasculature for increased xylem loading. The latter process is
accompanied by Zn distribution and detoxification in shoots, where metals are detoxified and stored in vacuoles with a
high storage capacity, as well as a phloem redistribution (Marschner, 1995). Both symplastic and apoplastic fluxes may
contribute to net Zn fluxes to the shoot (Sattelmacher, 2001; White et al., 2002).

Hyperaccumulator plants represent a low cost treatment for remediating metal polluted soils for their capacity to extract
high concentrations of metals and concentrate them in their upper parts. However, for achieving commercially
remediation programs, the bioconcentration factor (BCF, expressed as the ratio of trace element concentration in plant
shoot to trace element concentration in the soil) should be considered. Hence, the high Zn concentration in aereal tissues
and the higher the biomass production, the less the time necessary to reduce the pollutant content in the soil.

Ideal plants for phytoremediation purposes should be fast growing, have high a biomass, deep roots, be easy to harvest
and should tolerate and accumulate a wide range of trace elements in their aerial or harvestable parts. Native plants
should be preferred because they are better adapted in terms of survival, growth and reproduction than plants introduced
from other environment. To date, no plant has been described that fulfils all these criteria. Plant adaptation to high soil
trace elements availability usually include slow growth rates, and high root to shoot biomass ratio (Chaves et al., 2003). In
order to increase the chance of survival in adverse environmental conditions, plants priorize sexual reproduction at the
cost of reducing size and shortening the life cycle. This fact is considered a serious constraint to the use these plants for a
commercial-based phytoextraction programs. To overcome the low growth rate and low biomass production of
hyperaccumulator plants, different strategies can be considered. One of the most promising is the application of
biotechnology for adding specific traits from metal-hyperaccumulator to high biomass crops by using genetic engineering
methods.

5. Feasibility of plant biotechnological improvement for phytoremediation strategies

In general terms, there is an agreement that metal hyperaccumulation is an evolutionary adaptation proving protection
against herbivores and pathogens (Bhargava et al. 2012). Less than 0.2% of angiosperms have this capacity. This property
has been reported in over 450 species of vascular plants from 45 angiosperm families, including members of Asteraceae,
Brassicaceae, Caryophyllaceae, Cyperaceae, Cunonisceae, Fabaceae, Flacourtiaceae, Lamiaceae, Poaceae, Violaceae and
Euphorbiaceae (Bhargava et al. 2011). However, the best known angiosperm hyperaccumulator of metals is Thlaspi (now:
Noccaea) caerulescens (pennycress), that can accumulate large amounts of Zn (39,600 mg kg -1) and Cd (1800 mg kg-1)
without showing visual toxicity symptoms. Because of the easy growing under laboratory conditions, this plant has been
widely used in experimental system to study the mechanisms of Zn uptake, accumulation and tolerance in relation to Zn
phytoextraction. As most of the research background on this species has been made on a laboratory scale using hydroponic
systems, there is still a knowledge gap regarding Zn hyperaccumulation behavior under field conditions in contaminated
soils.

There was an impressive progress in characterizing soil chemistry management needed for phytoremediation and
physiology of metal-accumulating plants (Shao et al. 2010). Recent advances using novel methodologies (e.g. microarray-
based transcript analysis, Synchrotton X ray fluorescence microscopy, etc.) contributed to achieve a more comprehensive
global picture of plant metal homeostasis (Puig and Pearrubia, 2009). However, phytoremediation technology is still an
emerging technology, with only few practical or commercial applications.

The biochemical bases of metal accumulation was recently covered by other authors with great detail (Hall and Williams,
2003; Eide, 2003; Puig and Pearrubia, 2009; Lal, 2010; Hassan and Aarts, 2011; Claus and Chavarria-Krauser, 2012).

Most of the work on hyperaccumultors has focused on the physiological mechanism of metal uptake, transport, and
sequestration, but relatively little is known about its genetic bases (Lal, 2010). Genetic variations in the ability to
accumulate metals are a key point to be addressed for improving metal accumulation traits, either in conventional and
genetic engineering approaches. Following is described the state of the art and recent advances of genetic-based approaches
for enhancing metal accumulation features in plants:

5.1 Conventional breeding

Traditional breeding uses available genetic variability within species to combine the traits useful for phytoextraction
(Bhargava et al. 2012) and evolves both selection strategy (e.g. identification of diverse parental combination) and
transferring of metal tolerance/enhanced uptake phenotype traits from small, slow growing hyperaccumulator species to fast
growing and high biomass producing crops. This is achieved by hybridization strategies. Among breeding tools, symmetric
and asymmetric somatic hybridation bas become an increasingly used tool for genetic improvement. Moreover, in vitro
breeding and somaclonal variation have been used to improve the potential of Brassica juncea to extract and accumulate
toxic metals, with successful results. Beside of this case, little research has been carried out on breeding assessment of metal
accumulation trials in other high yielding species. Therefore, a research effort is needed for increasing scientific knowledge
on this matter. Mayor research needs should be focus on field studies, where a huge lack of information can be observed.

5.2 Genetic engineering

Conventional breeding deals with some severe sexual incompatibility constraints between taxa. For overcoming this issue
Biotechnology open new opportunities by direct transferring of specific genes to target plants. Thus, by applying genetic
engineering technology it can be obtained a transgenic plant that combine both enhanced metal accumulation features and
high biomass yield. Although scientific knowledge of molecular basis underlying metal tolerance/accumulation mechanism
has grown impressively over recent times, there limited information on metal perception and signal transduction pathways
in plants (Shao et al. 2010). Gene expression is a complex process evolving frequently different genes. Furthermore, metal
accumulation related genes have a tissue-depending expression, with a gradient of expression intensities depending on
genotype features, environmental conditions and their interaction. In other words, gene expression takes place at tissue
level, usually with a gradual modulation of the expression depending of genotypic to environment interactions. Heavy
metals affect plant gene expression at different scales, affecting DNA directly or via chromatin structure modification. The
activation of heavy metal stress-responsive genes occurs by a complex array of signaling pathways, which is a dimensional
network. Different secondary mediators participate in the activation of regulatory proteins that bind to promoter regions of
target genes and also posttranscriptional regulation occurs (Figure 1).
 Figure 1. A framework for the gene expression and regulation when plants are exposed to trace elements

The introduction and over expression of the hyperaccumulating genes into a non-hyperaccumulator plant could be a
promising strategy for enhance metal uptake, accumulation, tolerance and detoxification features (Bhargava et al 2012).
Many promising genes are reported to participate in these processes and different genetic engineering are used by use them
in transgenic plants (Table1 and Table 2).

Table 1. Some important genes involved in metal tolerance and accumulation (adapted from Bhargava et al. 2012)

Metal Gene
Al aha2
Fe frd3
Zn mtp1, mtp3
Cu Cbf
Cd atcax2, atcax 4, mt1
Ni reg2, sat-c

Table 2. Transgenic approaches for enhancement of Zn uptake in different plants (Adapted from Bhargava et al. 2012)

Plant Methodology/approach
Arabidopsis Overexpression of Zn Induced Facilitator 1 (ZIF1)
Brassica juncea Overproduction of the glutamylcysteine synthetase and glutathione synthetase
 Nicotiana tabacum Overexpression of glyoxalase pathway enzymes

Genetic engineering is a powerful technology for improving Zn accumulation traits of high biomass plants for
phytoremediation purposes. By applying this technology an ideal plant can be obtained combining both phyto-
accumulation traits (e.g. high metal accumulation) and high biomass productivity. A general model for developing a metal
tolerance transgenic plant is shown in Figure 2.

Figure 2. Development of metal tolerant/accumulator plant using genetic engineering (adapted from Karenlampi et
al. 2000, cited by Bhargava et al. 2012).

An ideal transgenic plant for phytoextraction purposes should have the following characteristics: tolerate high levels of
the element in roots and shoot cells, ability to traslocate the metal from roots to shoots, rapid metal uptake, high growth rate
and high aboveground biomass

Apart of metal accumulating genes, other kind of traits has been gaining research interest such as organic acid exudation
and siderophores exudation at rhizosphere level. By introducing genes encoding these phenotypic characteristics, an
improvement in metal uptake may be obtained due to the formation of metal chelating mechanisms.

Nowadays, the transgenic approach is an incipient technology only being tested at field scale for Hg, but environmental
issues has been stressed since Hg is volatilized at the soil surface and can eventually be re-deposited on soil or water
resources (Bhargava et al. 2012). In spite of this context, clonal propagating has opened new possibilities through tree
remediation cultivars (e.g. Populus x canescens), with huge metal accumulator features.

Some of main reasons behind the quite low applicability of phytoremedition technologies are as follows: long period
required for clean up the soil, limited number of target metals that can be extracted, limited depth that can be reached by the
root system, lack of knowledge on the agronomic and management practices to be applied under metal-contaminated soils
and scarce understanding of metal hyperaccumulator plants either at tissue or subcellular level

6. Conclusions and future perspectives

Plants have evolved different responses to deal with stress originated from high trace element availability in soils. In
particular, Zn-tolerant plants developed the following strategies to keep Zn homeostasis: Zn exclusion, Zn tolerant or Zn.-
hypertolerant species. These efficient processes include physiological, molecular, genetic and ecological traits which give
certain species the ability to survive in contaminated sites. A great deal of knowledge has been generated on Zn tolerance
and hyperaccumulation in the last years. Adapted plant response mainly depend on cells molecular mechanism and by a
series of signal transductions. Future research has to ensure a better understanding of these molecular mechanisms. On the
other hand, the use of hyperacumulators for phytoremediation technologies require a rapid metal uptake, high growth rate,
high aboveground biomass and a high rate of root-to-shoot metal transfer. The growing application of genetic technologies
may led to increase plant trace elements resistance strategies, as well as increased uptake of trace elements.

7. References

Adriano, D C. 2001. Trace elements in terrestrial environments: biogeochemistry, bioavailability and risks of metals, 2nd
edn., p. 860. Springer, New York.

Alloway BJ (1995). Heavy metals in soils 2nd edition. Blackie Academic Professional, London. P. 368.

Alloway, B.J. 2008. Zin in Soils and Crop Nutrition. International Fertilizer Industry Association (IFA). International Zinc
Association (IZA). 135 p.

Alloway, B.J. 2009. Soil factors associated with zinc deficiency in crops and humans. Environmental Geochemistry and
Health 31: 537-548.

Arnon, DI and PR Scout. 1939. The essentiality of certain elements in minute quantity for plants with special reference to
copper. Plant Physiology 14:371-375.

Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM .2006. The role of root exudates in rhizosphere interactions with plants
and other organisms. Annu Rev Plant Biol 57:233266

Baker AJM, Brooks RR 1989. Terrestrial higher plants which hyperaccumulate metallic elementsa review of their
distribution, ecology and phytochemistry. Biorecovery 1:81 126

Barak, P., Helmke, P. A. 1993. The chemistry of zinc. In A. D. Robson (Ed.), Zinc in soils and plants (pp. 90106). Dordrecht:
Kluwer Academic Publishers.

Barber SA. 1995. Soil nutrient bioavailability, 2nd edn. New York, NY, USA: John Wiley & Sons, Inc.

Bell PF, Chaney RL, Angle JS (1991) Free metal activity and total metal concentrations as indices of micronutrient
availability to barley [Hordeum-vulgare (L.) Klages]. Plant Soil 130:5162
Bhargava, A; FF Carmona; M Bhargava & S Srivasava. 2012. Approaches for enhanced phytoextraction of heavy metals.
Journal of Enviromental Management. 105: 103-120.

Broadley M, White PJ, Hammond JP, Zelko I, Lux A 2007. Zinc in plants. New Phytol. 173:677-702.

Brooks RR, Lee J, Reeves RD, Jaffre T.1977. Detection of nickeliferous rocks by analysis of herbarium species of indicator
plants. Journal of Geochemical Exploration7,4957.

Chaney R.L. 1993. Zinc phytotoxicity. A.D. Robson (Ed.), Zinc in Soils and Plants, Kluwer Academic Publishers, Netherlands
(1993), pp. 135144

Chaves M.M., J.P. Maroco, J.S. Pereira. 2003. Understanding plant responses to drought from genes to the whole plant.
Funct. Plant Biol., 30: 239264

Claus, J; A Chavarria-Krauser. 2012. Modeling regulation of zinc uptake via ZIP transporters in yeast and plant roots. Plos
ONE. Vol. 7. Issue 6. 11p.

Claus, J; A Chavarria-Krauser. 2012. Modeling regulation of zinc uptake via ZIP transporters in yeast and plant roots. Plos
ONE. Vol. 7. Issue 6. 11p.

Degryse F, Smolders E, Parker DR. 2006. Metal complexes increase uptake of Zn and Cu by plants: implications for uptake
and deficiency studies in chelator-buffered solutions. Plant Soil 289: 171185

Eide, DJ 2006. Zinc transporters and the cellular trafficking of zinc. Biochimica et Biophysica Acta 1763: 711-722.

Eide, DJ 2006. Zinc transporters and the cellular trafficking of zinc. Biochimica et Biophysica Acta 1763: 711-722.

Entry JA, Rygiewicz PT, Watrud LS & Donnelly PK. 2002. Influence of adverse soil conditions on the formation and function
of arbuscular mycorrhizas. Adv. Environ. Res. 7: 123138

Friedland, AJ. 1990. The movement of metals through soils and ecosystems. In: Shaw AJ, ed. Heavy metal tolerance in
plants: evolutionary aspects. Boca Raton, FL, USA: CRC Press, 719.

Garbisu C, Alkorta I .2003. Basic concepts on heavy metal soil bioremediation. Eur J Min Proc Environ Prot 3:5866

Guerinot ,ML .2000.The ZIP family of metal transporters. Biochim Biophys Acta. 1465: 190-8.

Hall, JL & LE Williams. 2003. Transition metal transporters in plants. Journal of Experimentary Botany. Vol. 54. N.
393:2601-2613

Hassan, Z; MGM Aarts, 2011. Opportunities and feasibilities for biotechnological improvement of Zn, Cd or Ni tolerance
and accumulation in plants. Enviromental and Experimental Botany. 72: 53-63

Hassan, Z; MGM Aarts, 2011. Opportunities and feasibilities for biotechnological improvement of Zn, Cd or Ni tolerance
and accumulation in plants. Enviromental and Experimental Botany. 72: 53-63

Holm, P.E., T.H. Christensen, J.C. Tjell, and S.P. McGrath. 1995. Speciation of cadmium and zinc with application to soil
solutions. Environ. Qual. 24:183-190.
Kabata-Pendias, A. 2001. Trace elements in soils and plants (3rd ed.). Boca Raton: CRC Press.

Lal, N. 2010. Molecular mechanisms and genetic bases of heavy metal toxicity and tolerance in plants. In: Plant Adaptation
and Phytoremediation. Capter 11. M. Asharf; M.Ozturk; M.S.A. Ahmad (eds). Springer. pp 227-244

Lasat MM, Baker AJM, Kochian LV. 1998. Altered Zn compartmentation in the root symplasm and stimulated Zn
absorption into the leaf as mechanisms involved in Zn hyperaccumulation in Thlaspi caerulescens. Plant
Physiology.118:875883

Lasat, M.M., 2002. Phytoextraction of toxic metals: A review of biological mechanisms. J. Environ. Qual., 31: 109-120.

Leyval, C., Turnau, K., Haselwandter, K., 1997. Effect of heavy metal pollution on mycorrhizal colonization and function:
physiological, ecological and applied aspects. Mycorrhiza 7, 139153.

Ma YB, Uren NC. 2006. Effect of aging on the availability of zinc added to a calcareous clay soil. Nutrient Cycling in
Agroecosystems 76, 11-18.

Marschner H. 1995. Mineral nutrition of higher plants, 2nd edn. London, UK: Academic Press.

Mason B., Moore, C.B., 1979. Principles of Geochemistry. John Wiley and Sons, Inc. New York, USA.

Maze, P. 1915. Determination des elements minerauxraresnecessaires au developpement du mals. C.R. Acad. Set., Paris,
160, 211-14.

McBride, M., Sauve, S. and Hendershot, W. 1997. Solubility control of Cu, Zn, Cd and Pb in contaminated soils. European
Journal of Soil Science. 48, 337-346.

McLaughlin MJ, Smolders E, Merckx R and Maes A (1997) Plant uptake of Cd and Zn in chelator-buffered nutrient solution
depends on ligand type. In: Ando T et al (eds) Plant nutritionfor sustainable food production and environment.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 113118

Meers, E., Unamuno, V.R., Du Laing, G., Vangronsveld, J., Vanbroekhoven, K., Samson, R., Diels, L., Geebelen, W., Ruttens,
A., Vandegehuchte, M., Tack, F.M.G. 2006. Zn in the soil solution of unpolluted and polluted soils as affected by
soil characteristics. Geoderma 136: 107-119

Neutelings G. 2011. Lignin variability in plant cell walls: contribution of new models. Plant Sci 181(4):379386

Nilsen, E.T., Orcutt, D.M. 1996. The Physiology of Plants under Stress: Abiotic Factors. New York, John Wiley & Sons. 689
pp.

NJDEP 1996. Soil Cleanup Criteria, New Jersey Department of Environmental Protection, Proposed Cleanup Standards for
Contaminated Sites, NJAC 7:26D.

Norvell, W.A. 1991. Reactions of metal chelates in soils and nutrient solutions. In Micronutrients in Agriculture. 2nd. edn.
(ed. Mortvedt, J.J., Cox, F.R., Shuman, L.M. and Welch, R.M.). pp. 187-227. Soil Sciene Society of America,
Madison.
Padmavathiamma, PK & LY Li. 2007. Phytoremediation technology: hyper-accumulation metals in plants. Water Air Soil
Pollut. 184: 105-126.

Palmgren MG, Clemens S, Williams LE, Krmer U, Borg S, Schjrring JK, Sanders D (2008) Zinc biofortification of cereals:
problems and solutions. Trends Plant Sci 13: 464473

Puig, S & L Pearrubia. 2009. Placing metal micronutrients in context: transport and distribution in plants. Current Opinion
in Plant Biology. 12: 229-306.

Reeves RD, Baker AJM . 2000. Metal-accumulating plants. In: Raskin I, Ensley BD (eds) Phytoremediation of toxic metals:
using plants to clean up the environment. Wiley, New York, pp 193229

Reichman S.M., Parker D.R. 2005. Metal complexation by phytosiderophores in the rhizosphere, In Biogeochemistry of
Trace Elements in the Rhizosphere, pp 129-156

Riley R. G., J. M. Zachara, and F. J. Wobber, 1992. Chemical contaminants on DOE lands and selection of contaminated
mixtures for subsurface science research, US-DOE, Energy Resource Subsurface Science Program, Washington,
DC, USA.

Sarret G, Harada E, Choi Y, Isaure MP, Geoffroy N, Fakra S, Marcus MA, Birschwilks M, Clemens S, Manceau A. 2006.
Trichomes of tobacco excrete zinc as zinc-substituted calcium carbonate and other zinc-containing compounds.
Plant Physiol 141:10211034

Sattelmacher B. 2001. The apoplast and its significance for plant mineral nutrition. New Phytologist 149: 167192.

Schalscha, E.B., Escudero, P., Salgado, P., Ahumada, I. 1999. Chemical forms and sorption of copper and zinc in soils of
central Chile. Communications in Soil Science and Plant Analysis 30: 497-507

Shao, H; L Chu; F Ni; & D Guo. 2010. Perspective on phytoremediation for improving heavy metal-contaminated soils. In:
Plant Adaptation and Phytoremediation. Capter 11. M. Asharf; M.Ozturk; M.S.A. Ahmad (eds). Springer. pp 227-
244

Shuman LM. 1991. Chemical forms of micronutrients in soils. In: Mortvedt JJ et al (eds) Micronutrients in agriculture, ASA,
CSSA, and SSSA. Madison, WI, pp 113144

Sinclair SA, Krmer U. 2012. The zinc homeostasis network of land plants. Biochimica et Biophysica Acta (BBA) - Molecular
Cell Research, 1823: 1553-1567

Sommer AL, Lipman CB. 1926. Evidence on the indispensable nature of zinc and boron for higher green plants.Plant
Physiology 1: 231249

Sytar, O., Kumar, A., Latowski, D., Kuczynska, P., Strzaka, K., Prasad, M.N.V. 2012. Heavy metal-induced oxidative damage,
defense reactions, and detoxification mechanisms in plants. Acta Physiologiae Plantarum , pp. 1-15

Torri S, Lavado R. 2008. Zn distribution in soils amended with different kinds of sewage sludge. Journal of Environmental
Management (Elsevier, Amsterdam, The Netherlands), 88: 1571-1579
Torri S, Lavado R. 2009. Plant absorption of trace elements in sludge amended soils and correlation with soil chemical
speciation. Journal of Hazardous Materials, 166: 14591465.

Torri S, Perez-Carrera A, Fernndez-Cirelli A. 2012. Zinc availability to forage crops in soils of the pampas region, Argentina.
In: Trace Elements: Environmental Sources, Geochemistry and Human Health. Editores: D. De Leon y P.R. Aragon,
Nova Science Publishers, Inc., Hauppauge, NY 11788

Torri S, Perez-Carrera A, Fernndez-Cirelli A. 2012. Zinc availability to forage crops in soils of the pampas region, Argentina.
In: Trace Elements: Environmental Sources, Geochemistry and Human Health. Editores: D. De Leon y P.R. Aragon,
Nova Science Publishers, Inc., Hauppauge, NY 11788

Von Wirn, N., Marschner, H., Rmheld, V. 1996. Roots of iron-efficient maize also absorb phytosiderophore-chelated
zinc. Plant Physiology 111 (4) , pp. 1119-1125

Wang P, Zhou DM, Luo XS, Li LZ . 2009. Effects of Zn-complexes on zinc uptake by wheat (Triticum aestivum) roots: a
comprehensive consideration of physical, chemical and biological processes on biouptake. Plant Soil 316: 177
192

Weissenhorn, I., Leyval, C., Berthelin, J. 1995. Bioavailability of heavy metals and abundance of arbuscular mycorrhiza in a
soil polluted by atmosphere deposition from a smelter. Biol. Fert. Soils 19, 2228.

Weng, L.P., Temminghoff, E.J.M., van Riemsdijk, 2001. Contribution of individual sorbents to the control of heavy metal
activity in sandy soil. Environmental Science and Technology 35, 44364443.

White PJ, Whiting SN, Baker AJM, Broadley MR. 2002. Does zinc move apoplastically to the xylem in roots of Thlaspi
caerulescens? New Phytologist 153: 201207.

Wolt, J.D. 1994. Soil Solution Chemistry: Applications to Environmental Science. John Wiley and Sons, New York.

Wong J.W.C., Li K.L., Zhou L.X., Selvam A. 2007, The sorption of Cd and Zn by different soils in the presence of dissolved
organic matter from sludge Geoderma, 137: 310-317

Wu, Q., Hendershot, W.H., Marshall, W.D., Ge, Y. 2000. Speciation of cadmium, copper, lead, and zinc in contaminated
soils. Communications in Soil Science and Plant Analysis 31: 1129-1144.