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Environmental and Experimental Botany 67 (2009) 10–22

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Environmental and Experimental Botany

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ualities in plant tolerance to pollutants and their uptake andranslocation to the upper plant parts

os A.C. Verkleij a,∗, Avi Golan-Goldhirsh b, Danuta Maria Antosiewisz c,ean-Paul Schwitzguébel d, Peter Schröder e

Institute of Molecular and Cellular Biology, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The NetherlandsThe Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, 84990, IsraelDepartment of Plant Morphogenesis, Institute of Experimental Plant Biology, Warsaw University, Miecznikowa str. 1PL – 02-096 Warszawa, PolandLaboratory for Environmental Biotechnology, EPFL, Station 6, CH-1015 Lausanne, SwitzerlandDepartment Microbe-Plant Interactions, Helmholtz Zentrum München, German Research Center for Environmental Health,

ngolstädter Landstrasse 1, D-85764 Neuherberg, Germany

r t i c l e i n f o

rticle history:eceived 19 January 2009eceived in revised form 1 May 2009ccepted 25 May 2009

eywords:eavy metalsrganic xenobioticslantsetoxification mechanismsptakeransport

a b s t r a c t

There is a duality in plant tolerance to pollutants and its response to the pollutants’ stress.On the one hand some plants, (hyper)tolerant to heavy metals, are able to hyperaccumulate these

metals in shoots, which could be beneficial for phytoremediation purposes to clean-up soil and water. Onthe other hand tolerant food crops, exposed to heavy metals in their growth medium, may be dangerousas carriers of toxic metals in the food chain leading to food toxicity. There is an additional duality in planttolerance to heavy metals and that is in food crops that are tolerant and/or hyperaccumulators, whichcould be used on one hand for phytoremediation, under controlled conditions and on the other hand forfood fortification with essential metals.

Similarly, plants are also exposed to a large number of xenobiotic organic pollutants. Because theygenerally cannot avoid these compounds, plants take up, translocate, metabolize and detoxify many ofthem. There is a large variability in tolerance (defence) mechanisms against organic pollutants amongplant species. This includes production of reductants but also scavenger molecules like ascorbate andglutathione and expression of the P-450 defence system, and superfamilies of the enzymes glutathione-and glucosyl-transferases. Again, with view to organic pollutants, plant detoxification mechanisms mightwell protect the plant itself, but produce compounds with some deleterious potential for other organisms.

In this review we discuss these dualities on the basis of examples of agricultural and ‘wild’ speciesexposed to metal contaminants (mainly Cd) and organic pollutants. Differences in uptake and translo-cation of various pollutants and their consequences will be considered. We will separately outline theeffects of the organic and non-organic pollutants on the internal metabolism and the detoxification mech-
anisms and try to indicate the differences between both types of pollutants. Finally the consequences andsolutions of these dualities in plant tolerance to pollutants will be discussed.
© 2009 Elsevier B.V. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112. Availability and toxicity of metal contaminants and organic pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.1. Metal contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.2. Organic pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

3. Uptake and transport of metal contaminants and organic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.1. Metal contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1. Common routes for transport of Fe and Cd . . . . . . . . . . . . . . . .3.1.2. Common route for transport of Zn2+ and Cd2+ . . . . . . . . . . . .

3.2. Organic pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +31 5987052; fax: +31 5987123.E-mail address: [email protected] (J.A.C. Verkleij).

098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2009.05.009

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

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4. Detoxification mechanisms of plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154.1. Metal contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4.1.1. (Hyper)tolerant wild plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154.1.2. Engineering of more efficient detoxification of metals in crop species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4.2. Organic pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175. Dualities in plant tolerance to pollutants: consequences and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19. . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Trace elements like Cd, Fe and Zn are easily taken up by plantsnd translocated to the shoots. Cadmium is considered as one ofhe most serious threats to man due to its long-term accumulationn the soil leading to food chain contamination. However, essential

etals, for example iron and zinc, when fed in excess, could be alsothreat to human health and well being due to the ability of a plant

o accumulate very high amounts of them without any detrimentalffects (Nriagu, 1989; Clemens, 2006).

Regarding organic pollutants the pesticide residue discussionhows abundantly clear where the danger potential of organic pol-utants lies to man. Many of the organic xenobiotics deposited onlant surfaces are generally characterized as micropollutants, dueo their small concentrations. However, despite this, they can exertoxicity or stress if they are able to penetrate from the root or leafurface into living tissues. This is not only true for classical herbi-ides (Coleman et al., 2002), but also for organic air pollutants andoly Aromatic Hydrocarbons (PAHs).

There is an inherent duality in plant tolerance to heavy met-ls and organic pollutants. Both types of compounds are at lowoncentrations beneficial for plants (essential metals, secondaryompounds, ROS as signals, etc.) and in this way beneficial for alliving organisms in the food chain. However, heavy metals andrganic xenobiotics exert damaging effects to plants as well at sim-

lar or higher concentrations depending on specific compound andlant species/genotype. Due to internal defence/tolerance mecha-
isms plants might well protect themselves, but could also produceompounds with some deleterious potential for other organisms,ncluding man. Finally plants hypertolerant to these compoundsnd accumulating them in high amounts could be beneficial foran by using these plants for phytoremediation technologies (phy-
Fig. 1. Dualities in benefit of, damage by and defence agai

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

tostabilisation, phytoextraction) and biofortification (Guerinot andSalt, 2001; Vassilev et al., 2004).

So, the dualities in benefit of and damage by these classes ofcompounds are applicable to the plant itself as well as to its use byman.

In Fig. 1 the dualities in benefit of, damage by and defenceagainst heavy metals and organic xenobiotics in plants are shownin detail.

Both, heavy metals and organic xenobiotics, will cause dam-age after uptake and transport into plant tissues. Both are activeon enzyme molecules and disrupt or alter their functions, andboth can cause oxidative stress. In addition, organic pollutants (andsome heavy metals) are known to promote DNA adduct formation.Defence reactions are centred around binding and chelating forHM, while conjugation is a major defence reaction against organics.In both cases, glutathione is a biomolecule of central importance.Dualities appear in the final fate of both pollutant classes: seques-tration and storage in the vacuole seems to be a common pattern.It has been observed, that some heavy metals act synergistic withorganic pollutants in causing stress and inhibiting detoxificationenzymes. In low concentrations, heavy metals as well as organicxenobiotics have been found to be beneficial for plants, becausethey stimulate ROS and induce resistance against other stressors.Besides providing options for the removal of pollutants from theenvironment by plant based methods in phytoremediation, thementioned reactions might also be of good use for humans, becauseessential metals can be (hyper)accumulated by the same mech-
anisms, or phenolic compounds might increase the contents ofnatural antiradicals and make plants healthier. In addition, knowl-edge generated by the study on the molecular mechanisms of aplant response to heavy-metals and organic pollutants is being usedfor the engineering of plants for the purpose of phytoremediation
nst heavy metals and organic xenobiotics in plants.

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nd also plants with improved nutritional values (“biofortifica-ion”).

The purpose of the present review is to highlight these dualitiesor both organic pollutants and trace elements, being essential ele-

ents or toxic metals. Recent advances in the following topics willhus be addressed for both organic xenobiotics and trace elements:1) availability and toxicity; (2) uptake and transport; (3) detoxi-cation mechanisms; (4) the use of the knowledge on these basicrocesses for biotechnological purpose. Finally some consequencesnd solutions will be suggested.

. Availability and toxicity of metal contaminants andrganic pollutants

.1. Metal contaminants

The soil composition and the availability of trace elements haveo be regarded as important as the soil/sediment physical chem-stry with the cornerstones of pH and oxygen contents. Any of thebove parameters will possibly release formerly bound metals inn unpredictable way. Here, we are often lacking data allowing thecaling up from the soil particle or flower pot perspective to a fieldystem.

Trace elements can be biologically essential like Fe, Mn, Cu,n, Ni, and Mo or non-essential: e.g. As, Cd, Pb, Ag, Hg, etc. Toxiconcentrations of heavy metals can exert primary and secondaryffects on plants at different integration levels (molecular, cellu-ar and tissue) depending on the metal(s) in question (Ernst et al.,992) Toxicity of Cd for instance is largely determined by its affin-ty for sulphydryl groups in proteins, peptides and amino acids.he behaviour of other trace elements (Zn, Ni) is on the contrary

argely based on a high affinity to oxygen and nitrogen-containingigands. The so-called “redox cycling” of redox active metals like Cu,e (oxidation–reduction of Cu2+/Cu+; Fe3+/Fe2+) or of As produceseactive oxygen species causing the peroxidative destruction ofiomembranes and cellular damage (Freedman et al., 1989). Somerace elements (especially Cd) have a strong influence on plantater relations (Barcelo and Poschenrieder, 1990).

The general symptoms of heavy metal toxicity at the whole plantevel are stunted growth, root growth inhibition, chlorosis, red-rownish colorization and necrosis. Non-essential toxic metals origh concentrations of essential elements can inhibit cell division,ell elongation and photosynthesis, and can cause water stress.

In order to cope with the toxicity caused by the different met-ls, various defence mechanisms have to be evolved at the differentntegration levels of the plant. At the cellular level protection of

etabolically active sites in the cell seems to be achieved by a morefficient cellular compartmentation of the metal surplus, espe-ially into the vacuole. The synthesis of metal-binding ligands toequester excess of metals is supposed to be a further protectionechanism. At the tissue level, allocation of excess of metals toetabolically less active tissues, such as epidermal cells has been

roposed (Vazquez et al., 1992). At the whole plant level it seemshat the metal surplus is highly allocated to deciduous organs at theime of senescence, and a limited amount is allocated to the seedsErnst et al., 2000).

.2. Organic pollutants

Today more than 100,000 different organic chemicals are dis-
ributed on the European market, and one third of them exceeduantities of one tonne per annum (European Chemical Substances
nformation System (ESIS), 2005: http://ecb.jrc.it/ and EEA 2007:tate of the environment report No. 1/2007, European Environ-ent Agency, Brussels). Most of them have been introduced for

perimental Botany 67 (2009) 10–22

the benefit of daily life, medicine, food production and indus-trial purposes, and a good proportion of these compounds lacknatural counterparts. The majority of these compounds have arather poor biodegradability. Hence, water resources, but also soilsbecome more and more contaminated with these man-made pol-lutants. Moreover, some of these obscure pollutants may possessthe undesirable capability of having estrogenic activity on varioushigher organisms. We have to face the problem that many of themwill create increasingly environmental problems in all regions ofour continent, and worldwide (Chaudhry et al., 2001). Amongstthem are well known pesticides, plasticizers, fuel additives, flameretardants, medicines, explosives, colours and fragrances. Indus-trial activities are thus major sources of water pollution. Industrialwastewater discharged into aquatic ecosystems either directlyor because of inadequate treatment can lower water quality byincreasing concentrations of xenobiotic pollutants, thereby caus-ing adverse effects on human health and undesirable changes inthe composition of aquatic biota.

Current literature presents clear-cut evidence that the avail-ability of organic compounds is governed on one hand by theirlipophilicity and on the other hand depends on organic matter con-tent of the soil under consideration (Behrendt and Brüggemann,1993; Schröder and Collins, 2002).

Some compounds form “bound” residues with organic matter orhumus particles in the soil. Thorough testing is required to establishdata bases to identify whether they are really “Dead dogs” or rather“Time bombs”. Besides, the nature and rooting pattern of the veg-etation will have great influence on the solubility of xenobiotics.Exuding up to 25% of the net carbon fixed during photosynthesisinto the rhizosphere, plants modify given soil–xenobiotic inter-actions in multiple ways (Siciliano and Germida, 1998). Phenolicsecondary plant products and aliphatic compounds may solubiliseorganic molecules in the vicinity of roots, and sugars or organicacids will stimulate growth of bacteria that might co-metabolizeorganic pollutants to reactive metabolites. It has been demon-strated that isoproturon is metabolized to plant available andreactive compounds in rhizosphere soil (Schuelein et al., 1996;Schroll and Kühn, 2004), and the bacterial conversion of Arochlors(defined mixtures of PCB) to reactive metabolites has been one ofthe early results of bioremediation studies (Pal et al., 1980).

3. Uptake and transport of metal contaminants and organiccompounds

3.1. Metal contaminants

To cope with changes in the availability of nutrients insoil solution, with a deficiency or an excess of elements (bothmicronutrients and non-essential ones), plants have developed reg-ulation of metal uptake and subsequent distribution/redistribution/storage/detoxification mechanisms within their bodies, and trans-port processes which are crucial for the maintenance of metalhomeostasis.

Based on the available data, there are several families of pro-teins involved in the transport of transition metals in plants. Tothe largest families belong: (i) influx transporter families such asNRAMP (natural resistance associated macrophage protein), ZIP(zinc-regulated transporter, iron-regulated transporter protein),YSL (yellow-stripe 1-like subfamily of the OPT superfamily); and(ii) efflux protein families: P1BATPases, CDF (cation diffusion facil-itator), CAX (cation exchanger) and ABC (ATP-binding cassette
transporters) (Hall and Williams, 2003). Each transporting proteindoes usually catalyse the transport of several ions, however, withdifferent affinity.
Recently, a comparison of the substrate specificity of transport-ing proteins has revealed an interesting difference between influx

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nd efflux transporters (Krämer et al., 2007). An even more pro-ounced specificity has been found in those which export metals

rom the cytoplasm whereas proteins mediating metal influx (thuslso uptake from the soil/medium), for example ZIP and NRAMP,ppears less selective. Hence, the import of a variety of ions isess tightly controlled. It probably ensures the supply of all ionsecessary to perform a variety of functions by different cells and tis-ues; however, as a consequence non-essential ones are more easilybsorbed as well. The majority of the information on metal trans-ort processes comes from the study on Arabidopsis and the hyper-ccumulator species A. halleri and Thlaspi caerulescens and amongrop species rice is the best recognized plant (Krämer et al., 2007).

Plants do not discriminate well between micronutrients andon-essential toxic ions, and therefore take up and accumulate toifferent levels both Zn and Cd present in soil. Depending on thepecies-specific uptake system, and subsequent cell/tissue/organistribution of elements, their content and distribution is greatlyiversified (Greger, 2008; Prasad, 2008). Within crop species a largeenotypic variation exists in Cd distribution. Maize inbred lines fornstance can be divided in ‘shoot Cd excluders’ and ‘non-shoot Cdxcluders’ based on its partitioning in the plant. Cadmium concen-rations in the xylem exudates from ‘non-shoot Cd excluders’ areigher than those from ‘shoot Cd excluders’ (Florijn et al., 1993).imilar patterns are found in other crops like lettuce and tobaccoJarvis et al., 1976).

This differential Cd distribution between ‘shoot Cd excluders’nd ‘non-shoot Cd excluders’ may be related to the observed dif-erences in root Cd concentration, desorption characteristics andinding capacity of Cd inside and/or outside the root and its distri-ution within the roots (Florijn and Van Beusinchem, 1993).

The ability to take up toxic non-essential metals by crop plantseads to food contamination (Greger, 2008; Marmiroli and Maestri,008). Understanding the basic molecular mechanisms governinghese processes might pave the way to engineering of crop plantsble to discriminate between essential and non-essential elements,nd enable the production of plants with desirable metal contentn chosen organs.

Nutrient ions are transported by proteins belonging to a rangef families. However, according to current knowledge, plants doot possess specific transporters for non-essential metals likeadmium, and its transport across a membrane is mediated byystems for essential cations. According to available data, Cd2+ isransported by Fe2+, Zn2+ and Ca2+ transporters or channels ofroad substrate specificity (Krupa et al., 2002; Clemens, 2006;ntosiewicz et al., 2008). A wide range of mutual interactions haseen shown between these ions. Over the course of numerous soilnd hydroponic experiments, it has been demonstrated that cad-ium interacts with nutrients, interfering often in their uptake,

istribution at the plant level, causing nutritional deficiencies, etc.Krupa et al., 2002). Little by little, the molecular study of the trans-

embrane transport of metals reveals the mechanisms underlyinghese phenomena.

In non-hyperaccumulating (hyper)tolerant plant species theres little evidence for an avoidance mechanism with respect to Cdnd Zn: the total uptake of Zn or Cd is not significantly differentetween sensitive and hypertolerant populations of Silene vulgaris

n short-term experiments (Harmens et al., 1993a; De Knecht,994), indicating that an internal sequestration mechanism muste operative in hypertolerant plants.

In hyperaccumulating plants however it has been clearly shownhat the Zn uptake in T. caerulescens is much higher compared with
. arvense (a non-hyperaccumulator species).
This difference is due to a higher Vmax in T. caerulescens whilehe Km values appear similar for both species (Lasat et al., 1996).hese data suggest a higher expression of functionally very similarn transporters in T. caerulescens compared to T. arvense roots (Lasat

perimental Botany 67 (2009) 10–22 13

and Kochian, 2000). Molecular expression studies on Zn trans-porters have confirmed these results: ZNT1/ZNT2, members of theZIP family of metal transporter genes are mainly expressed in rootsof T. arvense, but only under Zn deficiency conditions. At normal orelevated Zn supply their transcription is strongly down regulated(Grotz et al., 1998; Pence et al., 2000; Assuncão et al., 2001). By con-trast, in T. caerulescens both ZNT1 and ZNT2 are highly expressed inroots under low and high Zn supply.

The mechanism of Cd uptake might be different from Zn uptakein T. caerulescens. Kinetic studies have clearly shown a difference inVmax of the saturable component, which is about 5 times higher inthe Ganges population than in the population from Prayon (Lombiet al., 2001; Zhao et al., 2002). There is also a clear interferencebetween the Cd and Fe uptake in the Ganges population, whichdoes not appear in the Prayon population (Roosens et al., 2003).Furthermore Ca uptake in the Prayon ecotype is inhibited by theaddition of Cd, which leads to the suggestion that at least two otheruptake systems must be involved in Cd uptake (Roosens et al.,2003).

3.1.1. Common routes for transport of Fe and CdMetal transporters regulated by iron can mediate the traffick-

ing of a variety of divalent cations, including cadmium. It hasbeen reported that plants grown under Fe-limiting conditions takeup and accumulate more Cd (Cohen et al., 1998; Colangelo andGuerinot, 2006). First, the study on A. thaliana revealed that AtIRT1,the broad range transporter from ZIP family, up-regulated underFe-deficiency and primarily responsible for Fe uptake into the rootepidermis, can also transport other metal ions like Mn2+, Zn2+

and Cd2+ (Eide et al., 1996; Rogers et al., 2000) and is responsi-ble for enhanced Cd accumulation. Hence, a plant strategy for moreefficient recruitment of nutrients results in more intensive accu-mulation of unwanted Cd and contamination of crops. Similar toAtIRT1, other ZIP proteins from rice, Fe2+ transporters OsIRT1 andOsIRT2, are also involved in the uptake and translocation of Cd,especially under Fe-deficiency. Expression and tissue localizationstudies suggest that they are responsible for both the uptake ofCd and its distribution throughout the plant body (Ishimaru et al.,2006; Nakanishi et al., 2006). Although IRT genes have been clonedfrom several plant species, in the first place the transport of iron hasbeen studied, and only for some of them experiments have beenperformed to check if they exhibit broad substrate specificity. Forexample, although LeIRT1 and LeIRT2 from Lycopersicon aesculentumhave been cloned, nothing is known so far about their involvementin the translocation of other cations than iron (Bereczky et al.,2003).

The existence of transporting proteins mediating the translo-cation of several ions raises the question of the nature of thisbroad specificity, accompanied by distinct affinity for different ions.Rogers et al. (2000) in their study on AtIRT1 have shown thatreplacement of key aspartate residues (Asp-100 and Asp-136) withalanine, converts the transporter into a form that retains the abilityto transport fully only zinc, and at a reduced rate cadmium, whileiron and manganese translocation is abolished. The results indi-cate that transport mechanisms of different metals are physicallyseparable, and also that substrate specificity involves more thanjust a few key amino acids. Full understanding of the mechanismof the control of substrate specificity would enable the produc-tion of mutated forms of proteins and, for example, with their use,possibly engineering of plants able to take up efficiently nutrientsand eliminating/reducing cadmium (or other toxic metals) in crop
species.
NRAMP is another family of proteins which, in addition to thecontribution to Fe homeostasis (Grotz and Guerinot, 2006), areimplicated in the transport of cadmium. For example, NRAMP1from A. thaliana is able to transport primarily Fe2+ but also Cd2+ and

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n2+ (Curie et al., 2000; Thomine et al., 2000). Up-to-date resultsndicate that in crop species some NRAMPs are also involved inadmium uptake. Several members of this family have been iso-ated from rice and tomato (Belouchi et al., 1997; Bereczky et al.,003), and very recently, the involvement of NRAMP proteins inhe cadmium transport has been shown by Xiao et al. (2008). Theyave demonstrated by heterologous complementation that MbN-AMP1, iron-regulated transporter from Malus baccata expressedainly in roots, is involved in Fe2+ and Mn2+ transport, but also in

d2+ trafficking.

.1.2. Common route for transport of Zn2+ and Cd2+

In general, ZIP family is one of the most important group ofroteins in mediating the uptake of cations, mostly zinc and iron,owever, it has been demonstrated for several proteins from dif-

erent plant species (including Arabidopsis and crop plants) thathey are able to take up cadmium as well (Hall and Williams,003; Colangelo and Guerinot, 2006). In crop plants, for exampleice, there are 17 ZIP-coding genes identified (Chen et al., 2008).ut of them, OsZIP1, OsZIP3 and OsZIP4 are associated primar-

ly with Zn transport (Ramesh et al., 2003). The yeast expressionxperiments as well as expression and localization study in riceed to the conclusion that OsZIP1 with inducible gene expres-ion (not detectable under control conditions, but induced by Zneprivation) exhibits broad substrate specificity being permeableo cadmium and other cations, whereas OsZIP3 is more selectiveor zinc, and its expression (both in roots and shoots) is more con-titutive. Kinetic study of yeast cells expressing rice cDNAs OsZIP1nd OsZIP3 has demonstrated similar affinity for zinc, but the Vmax

or uptake is 2-fold higher for cells expressing OsZIP1. The pres-nce of cadmium reduces the growth of the yeast strain expressingsZIP1 but not OsZIP3. The localization of transcript of both genes

o the vascular tissue suggests a role for zinc absorption (and otherransported cations) or transfer from the vascular tissue. On thether hand, OsZIP4 encoding a Zn transporter (its possible involve-ent in the uptake of cadmium was not tested) localized to the

lasma membrane is highly expressed in roots and shoots undern-deficiency and under such conditions its transcript is morebundant than those of OsZIP1 and OsZIP3. Its protein is localizedo the plasma membrane and expression is mostly found in phloemells in roots and shoots and in the meristem (Ishimaru et al.,005).

Furthermore, the study performed by Moreau et al. (2002) indi-ates the possibility of the involvement of another ZIP protein fromhe crop plant Glycine max, primarily in the transport of zinc, and inddition cadmium. It has been shown that GmZIP1 is highly selec-ive for zinc with Km value of 13.8 �M, and cadmium is the only

etal able to inhibit Zn uptake in transformed yeast.However, it has to be underlined that ZIPs are considered as one

f the major class of proteins involved in Zn uptake in plants, andp-to-date only some of them have been shown to be able to trans-ort cadmium (Colangelo and Guerinot, 2006; Grotz and Guerinot,006). The determination of the in planta expression profile hasevealed that ZIP genes from rice, tomato or soybean are differen-ially regulated in different tissues and organs, some of them inesponse to Zn status, some of them constitutively. In general, theyre likely involved in a very complex metal homeostasis, and theirhysiological role is to be established in the future.

CDF is the next class of transporting proteins catalysing thefflux of divalent cations, and Zn2+ is one of their major sub-trate, besides Co2+, Fe2+, Cd2+, Ni2+ and Mn2+ (recent review by
ontanini et al., 2007; Antosiewicz et al., 2008). The best charac-
erized CDFs (renamed MTPs – Metal Tolerance Proteins) originaterom A. thaliana. Its overexpression confers Zn-tolerance in planta.rabidopsis plants overexpressing the transporter ZAT becomeore Zn2+ tolerant and accumulate more Zn (Van der Zaal et al.,


1999). AtMTP1 is localized to the vacuolar membranes of leaf androot cells, suggesting a role in Zn sequestration in the vacuole(Dräger et al., 2004). These data suggest that a MTP like gene inZn hypertolerant plants could be involved in the more efficient Zntransport across the tonoplast in the root tissue, but so far molecu-lar evidence is lacking for tolerance and accounting for high MTP1transcript levels.

Crop plants are not covered by an extensive study so far. Yet, it isonly recently that Shingu et al. (2005) have isolated NgMTP1 fromNicotiana glauca and NtMTP1a and NtMTP1b from N. tabacum whichheterologously expressed in yeast mutants complements Zn- andCd-tolerance, hence suggesting that they might operate by seques-tering Zn and Cd into vacuoles.

There is also a very important P1BATPase (HMA) family oftransporters mediating the efflux of a range of transition metalsincluding Zn2+ and Cd2+ (Hall and Williams, 2003). It is known thatrice contains nine P1BATPases, however, not much has been dis-covered about their function (Baxter et al., 2003). Only recently amore detailed characteristic of HMAs from rice has been performed(Lee et al., 2007). Three HMAs (OsHMA5, OsHMA6, OsHMA9) are up-regulated by increased Zn and Cd concentrations. More detailedexamination of OsHMA9 has revealed its plasma membrane local-ization and involvement in Cu2+, Zn2+, Pb2+ (and possibly Cd)transport/detoxification. The mutant oshma9 accumulates more Cu,Zn, Pb and Cd and is more sensitive to elevated levels of Cu2+, Zn2+

and Pb2+ suggesting its role in the export of these cations from thecell.

3.2. Organic pollutants

Depending on the localization and depth of a given pollutionplume, not all plants in a given canopy will be in contact withorganic xenobiotics. Deep rooting species and plants with tap rootswill access pollutant plumes that have already moved to deeper soilzones, whereas surface pollution might be easily accessible to shal-low rooting plants. Here, differences between annual and persistingspecies will become important (Schnoor et al., 1995; Simonich andHites, 1995; Newman et al., 1997).

In the case of historic pollution, where the chemicals have agedin the soil, or had been complexed with minerals or organic mat-ter, the rooting pattern of the plant under consideration is evenmore important for potential uptake and accumulation of foreigncompounds and their metabolites in the upper plant parts. Thismay be a major constraint for growing deep rooting food cropson historic plumes, and decisive for the success of any envisagedphytoremediation; plants with a dense root system will be idealfor such a purpose and mobilize a significant proportion of thepollutant.

In case where direct contact has been established between theplume and the root surface, the transfer of the xenobiotic from thesoil or water to the plant is crucial. It is expected to proceed spon-taneously and diffusion driven for compounds with lipophilicityclose to that of the roots of given species. Uptake and transportof organic xenobiotics in roots has been reviewed by a number ofauthors (Briggs et al., 1982; Briggs and Bromilow, 1983; Behrendtand Brüggemann, 1993; Schröder and Collins, 2002). Furthermore,uptake of lipophilic and amphiphilic compounds from differentmedia has been intensively studied in the context of pesticideresearch. Data for the determination of the so-called root concen-tration factor, RCF, are available in the literature. The RCF describesthe potential of a given xenobiotic to accumulate in the plant root,
without differentiating between surface accumulation and uptakeinto the root tissue (Schröder and Collins, 2002). However, as theRCF is heavily dependent on the log KO/W, i.e. the lipophilicity of thecompound under consideration, it seems to yield a good estimateof the absorptive properties of the rhizodermis.

nd Ex

b

l

lbtim

flbafbnnztCc

ptoh

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4

4

4

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Briggs et al. (1982) have reported the following relationship forarley:

og (RCF − 0.82) = 0.77 log Kow − 1.52

Compounds with a low Kow (i.e. <1) will hardly penetrate theipid containing root epidermis, whereas compounds of log Kow > 3ecome increasingly retained by the lipid in the root epidermis andhe organic matter surrounding the root as a result of their increas-ng hydrophobicity (Schröder and Collins, 2002). In this case, uptake

ight be minute, if detectable at all.Uptake and translocation of various organic pollutants also dif-

er among plant species. Uptake may here depend on the pollutant’sipophilicity and dissociation constant (Schröder and Collins, 2002),ut also heavily on species inherent properties of the root itselfnd the transport tissues involved. Sulphonated anthraquinonesor instance are more efficiently translocated and metabolisedy Rheum (rhubarb) and Rumex (sorrel), plant species producingatural anthraquinones, than by maize or celery, anthraquinoneon-producing species (Aubert and Schwitzguébel, 2004). Shoots ofucchini (Cucurbita pepo L.) accumulate various hydrophobic con-aminants (i.e. DDE or dioxin) from soil (Campanella and Paul, 2000;ampanella et al., 2002), but many other plants do not, includingucumber (Cucumis sativus).

The uptake into the hydraulic system of the plant and thus theath into stem and leaves may be quantified by calculating theranspiration stream concentration factor, TSCF. Here, compoundsf intermediate solubility, weak acids and amphiphilic substancesave advantages facilitating their transport.

Compounds with a log Kow ∼ 2 are transported solely in the tran-piration stream, while those with a log Kow ∼ 1 are both, phloemnd xylem mobile, although it is probable that only metabolitesnter the phloem. For compounds with log Kow between 1 and 3etabolism may occur in the leaf and stem tissue and there maybe

elease to the atmosphere through leaf tissue, additionally “bound-esidue” can be created in the plants (Langebartels and Harms,986).

The processes of xylem-loading of foreign compounds and dis-ribution in leaf tissue have not been well investigated but arehought to be analogous to herbicide movement in a given plant.t might be assumed that metabolism of xenobiotics in plants isonfined to root and leaf tissues, and is only scarcely taking placeuring transport in the plant vascular system.

. Detoxification mechanisms of plants

.1. Metal contaminants

.1.1. (Hyper)tolerant wild plantsHypertolerant non-hyperaccumulating ecotypes of S. vulgaris

etain a higher fraction of Zn or Cd taken up in their roots com-ared with non-tolerant ecotypes (Harmens et al., 1993a; Denecht, 1994). Non-accumulator plants do not seem to have strongetal detoxification mechanisms in the shoots: preliminary results

uggest that non-hyperaccumulator plants also do not possessypertolerance mechanisms in the shoots (Chardonnens, 1999).ence most of the metals are excluded from the shoot via root

equestration, most likely to protect the photosynthetic apparatusn leaf cells which is extremely sensitive to heavy metals (Küppert al., 2000).

On the other hand, hyperaccumulator plants very actively
ranslocate heavy metals into the shoots, presumably because ofhe existence of heavy metal tolerance mechanisms operating inhe shoot. The non-accumulating ecotype of Sedum alfredii retainsearly 3 times more Zn in the root vacuoles than the hyperac-umulating ecotype (Yang et al., 2006). Moreover higher metal

concentrations have been found in the xylem sap of hyperaccumu-lators, due to enhanced xylem loading (Lasat et al., 1998). A numberof transporters seem to be involved in this process, under whichP-type ATPase-HMA, MATE (a large family of multi-drug and toxiccompound efflux membrane proteins) and OPT (oligopeptide trans-porters) and their possible involvement has been recently discussed(Verbruggen et al., 2009).

4.1.1.1. Vacuolar sequestration. There is some evidence that vacuo-lar compartmentation could be the basic mechanism concerning Znand Cd hypertolerance. A large fraction of Zn and Cd is located in thevacuole of sensitive plant species (Vögeli-Lange and Wagner, 1990)and in the Zn hyperaccumulator plant T. caerulescens zinc is foundmostly in the vacuole (Vazquez et al., 1992). Direct evidence comesfrom Zn uptake studies in Mg-ATP energized tonoplast vesicles iso-lated from Zn hypertolerant and sensitive S. vulgaris (Verkleij et al.,1998). Zn (as Zn-citrate) is more effectively transported over thevacuolar membrane in the Zn-tolerant ecotype and, based on plantcrosses, it has been established that Zn hypertolerance is genet-ically correlated with enhanced tonoplast transport of the metal(Chardonnens et al., 1999). Although AtMTP1 belonging to the CDFfamily seems to be a good candidate with respect to enhanced Zn-tolerance and accumulation, several other families of transporterscould be involved in the vacuolar sequestration of transition met-als. In addition to CDFs, there is evidence that HMAs, Ca2+/cationantiporter (CaCA) super family and the ABC (ATP-binding cassette)transporters also play a role in this sequestration process (see recentreview of Verbruggen et al., 2009).

4.1.1.2. Detoxification by trace metal-binding molecules. Once insidethe cytosol metal ions can be chelated by different ligands suchas organic acids, amino acids, peptides and proteins. The mostimportant ligands, which seem to play a role in hypertolerance and(hyper)accumulation are: glutathione (GSH), phytochelatins (PCs),metallothioneins (MTs), histidine (His), nicotianamine (NA) and theorganic acids malate and citrate. We will briefly summarize the pos-sible role of these ligands and for more detailed information werefer to a recent review of Verbruggen et al. (2009).

GSH (�Glu-Cys-Gly) is a major cellular anti-oxidant, which canbind several metals and is a precursor of PCs. It might be pointedout here that GSH is also the co-substrate for the conjugation ofmany organic xenobiotics. Recent results with Ni and Cd suggestthat GSH could play a role in hyperaccumulation, probably due toits anti-oxidant activity.

PCs are derived from GSH by the action of a �-glutamylcysteinyltranspeptidase, phytochelatin synthase (PCS) (Grill et al., 1989).Because Cd2+ has a high affinity for thiolates, like many other toxictransition metals, it is mostly bound by these ligands and is notpresent as hydrated ion. PCs play a role in metal detoxification. Forinstance, mutants of A. thaliana, which are either glutathione or PCdeficient, are Cd sensitive (Howden et al., 1995). However in nat-urally selected Zn and Cd hypertolerance its role is questionable.De Knecht et al. (1992) and Harmens et al. (1993b) have clearlydemonstrated that increased production of PCs is not the mecha-nism underlying naturally selected hypertolerance to Cd or Zn in S.vulgaris.

MTs are small cysteine-rich proteins that can effectively bindmetals through thiolate cluster formation. Their functions havebeen implicated in copper homeostasis in A. thaliana (Murphy andTaiz, 1995). In Silene paradoxa and S. vulgaris, Cu hypertolerancehas been found to be associated with constitutively enhanced tran-
script levels of a 2b-type metallothionein gene, SvMT2b (Van Hoofet al., 2001; Mengoni et al., 2003). The role of MTs in Zn and Cdhypertolerance is probably very small (Jack et al., 2007). MTs of thetypes 1, 2 and 3 seem to function in Cu accumulation and phloemCu transport (Guo et al., 2008).

1 nd Ex

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His is the most important amino acid ligand involved in hyper-ccumulation and forms complexes with Ni, Cd and Zn. It has beeneported that Ni hyperaccumulation in Alyssum lesbiacum is dueo Ni complexation by histidine (Krämer et al., 1996). Results withther species of the Brassicaceae family suggest that His-dependenti xylem loading may not be universal (Verbruggen et al., 2009).

Organic acids malate and citrate: due to the low associationonstants with trace metals the role for organic acids in hyperac-umulation is not very likely (Callahan et al., 2006). It is suggestedhat these organic acids could play a role in vacuolar sequestration.

NA forms complexes with most transition metal ions and its roles supposed to help the movement of micronutrients through thelant. There is circumstantial evidence that NA could be involved

n metal hyperaccumulation in A. halleri and in T. caerulescensVerbruggen et al., 2009).

.1.2. Engineering of more efficient detoxification of metals inrop species

In principle, it is not desirable to grow crop species withnhanced ability to detoxify heavy metals; however, their highiomass, and in certain cases relatively high constitutive toler-nce to metals and accumulation, draws attention of scientists toquip such plants by genetic manipulation with the mechanism ofven more efficient detoxification for biotechnological purpose. Inngineering plants for phytoextraction focus is on enhancement ofptake, root-to-shoot translocation and detoxification capacity. Onhe other hand, molecular breeding plants more suitable for phy-ostabilization requires modifications resulting in increased metalptake, improved ability to store them in roots and reduced translo-ation to shoots. Another challenging task (called biofortification)s engineering of plants with improved nutritional quality of theirdible parts (roots, shoots, leaves, grains – depending on a croplant) by increase of their microelement content and decrease ofoxic heavy-metals (Kärenlampi et al., 2000; Grotz and Guerinot,002; Eapen and D’Souza, 2005; DalCorso et al., 2008).

Detoxification involves a specific link between two processes:mmobilization of metal ions and sequestration in cellular com-artments of lower than the cytoplasm metabolic activity which

nclude the vacuoles and the cell wall. The capacity of these pro-esses may be one of the crucial factors limiting the efficiency ofetoxification. To improve it, a strategy has been designed basedn transferring the genes coding for transport proteins involved inetal compartmentalization or those involved directly or indirectly

n the immobilization of metal ions. Most frequently Nicotiana andrassica species are used for the transformation.

.1.2.1. Engineering more efficient detoxification by immobilization.or engineering plants with enhanced metal detoxification capac-ty, it is expected that the introduction of genes for synthesis of

etal chelators to crop plants would improve their ability to immo-ilize metal ions, hence increase the level of tolerance, and possiblyptake and accumulation.

For transformation, genes coding enzymes from the phy-ochelatin biosynthesis pathway have been frequently used. Theverexpression of the last enzyme, phytochelatin synthase (PCS),epending on the gene and on species used for transformation,ives distinct results – decrease/increase of the Cd-accumulationnd decrease/increase of Cd-tolerance level (Li et al., 2004; Pomponit al., 2006; Wawrzynski et al., 2006; Wojas et al., 2008). Gasic andorban (2007) have attributed the enhanced Cd- and Zn-tolerance

n Indian mustard overexpressing AtPCS1 to the moderate increase
f the transgene expression. However, the most successful studiesre those of Gisbert et al. (2003) and Martínez et al. (2006). Theyave overexpressed TaPCS1 in N. glauca, a plant with a biomass 100imes higher than Cd/Zn hyperaccumulator T. caerulescens. Plantsested both in hydroponics and in contaminated soil exhibit greatly

enhanced accumulation of Cd, Pb and Zn in shoots and the levelof tolerance to metals. Transgenic tobacco grown in soil containingover 11,000 mg kg−1 of Pb and 4500 mg kg−1 of Zn yields an averageof 0.5 kg per plant while T. caerulescens is unable to survive.

For genetic modifications, enzymes from earlier steps of PCbiosynthesis have also been used – gshI (�-glutamylcysteinesynthetase), an enzyme controlling GSH synthesis, and gshII (glu-tathione synthetase). Their overexpression in Brassica juncea andNicotiana tabacum results in the increase of both GSH and PCs,what is accompanied by an increase of shoot Cd accumulation andtolerance (Zhu et al., 1999a,b; Wawrzynski et al., 2006).

Furthermore, transgenic Nicotiana or Brassica plants withenhanced phytochelatin and glutathione levels through overex-pression of cysteine synthase (Harada et al., 2001), or APS –adenosine triphosphate sulphurylase (Bennett et al., 2003) exhibithigher Cd-tolerance and accumulates significantly more Cd in theshoots than the wild-type plants.

The overexpression of metallothionein genes has not been verysuccessful in the generation of plants with improved accumulationor tolerance, and in changes of the distribution with more metal inthe harvestable plant parts. The best improvement has been madeby the transfer of yeast CUP1 gene to cauliflower which has resultedin 16-fold increase of Cd-tolerance and accumulation (Hasegawa etal., 1997). However, in most cases, metal accumulation/uptake intransgenic plants is not significantly altered. For example, introduc-ing HsMTII into tobacco does increase Cd-tolerance and alters themetal distribution pattern with more metal in roots (but withoutchange in the total metal per plant) (Elmayan and Tepfer, 1994).In general, the overexpression of MT genes from various organ-isms (human, mouse, yeast) in Nicotiana or Brassica increases theefficiency of the detoxification of cadmium (Cd-tolerance levelincreased) and alters the distribution (often the concentration ofthe metal increases in roots), but uptake is not much differentfrom the wild-type plants (Misra and Gedamu, 1989; Yeargan etal., 1992).

Alternatively, the increase of the nicotianamine concentra-tion has been engineered by the overexpression of nicotianaminesynthase (NAS) genes. For example, NAS1 from Hordeum vulgaretransferred to tobacco (Takahashi et al., 2003) has resulted inenhanced accumulation of Zn, Fe, Cu, Ni and Mn in shoots andthe level of tolerance to metals. Interestingly, in transgenic plantsgrown on enriched soil the content of Zn, Fe, Cu and Mn in seedsincreases by 3.2-, 6.6-, 2- and 1.8-fold, respectively.

4.1.2.2. Engineering more efficient detoxification by compartmental-ization. An attempt to improve the transport processes responsiblefor trafficking of metal ions to the cellular storage compartmentshas been made by the overexpression in tobacco calcium exchangeantiporters, AtCAX2 and AtCAX4, from Arabidopsis thaliana whichmediate with different affinity the transport of Ca2+, Cd2+, Zn2+

and Mn2+ to the vacuole (Hirshi et al., 2000; Korenkov et al.,2007a,b). Transformed plants are more tolerant to Cd, Zn and Mn,and exhibit 4-fold increase of Cd and 2-fold increase of Mn con-tent in roots. Furthermore, directly enhanced Cd- and Zn-selectivetransport into root tonoplast vesicles has been shown, indicatingimproved vacuolar sequestration due to the transformation, andlink higher storage capacity with observed enhanced level of tol-erance. On the other hand, the overexpression of the broad rangecation transporter from wheat TaLCT1 (Clemens et al., 1998) intobacco has resulted in increase of the metal tolerance (Cd and Pb),however with no spectacular alteration in the level of accumula-
tion (Antosiewicz and Hennig, 2004; Wojas et al., 2007). Hence, theimprovement of the detoxification does not always stimulate theuptake.
Another study has demonstrated the engineering of the distri-bution of Zn in rice plants by overexpressing a Zn transporter OsZIP4

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Akuttitth(ati

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nder the control of the cauliflower mosaic virus (CaMV) 35S pro-oter. The metal concentration in roots of transgenic plants is 10

imes higher than in those of vector controls, but it is 5 times lessn shoots. The Zn concentration in seeds is 4 times lower compared

ith control plants (Ishimaru et al., 2007). Hence, OsZIP4 is not rec-mmended for the purpose of biofortification to generate a plantith improved content of this micronutrient in seeds.

In a different approach, specific proteins mediating the transportf metal-conjugates, for example metal complexes with phy-ochelatins or glutathione into the vacuoles, have been used forhe heterologous overexpression.

These have been achieved by transferring to tobacco ScYCF1, theBC-family protein from yeast, belonging to the MRP-subfamilynown to pump Cd2+ conjugated to thiol compounds into the vac-oles (Plaza and Bovet, 2008). The resulting transgenics grow betterhan wild-type plants in Cd/Zn contaminated soil compared tohe wild-type, and exhibit enhanced accumulation of both metalsn leaves. On the other hand, the expression of human hMRP1 inhe same species improves the detoxification rendering higher Cd-olerance likely by more active sequestration of Cd in the vacuoles;owever, the accumulation level remains at that of the wild-typeYazaki et al., 2006). Furthermore, the increase of Cd-tolerancend Cd accumulation, enhanced retention in roots resulted fromhe overexpression of AtMRP7 also in tobacco (Wojas et al.,n press).

In conclusion, the performance of transgenic plants with mod-fied either transport processes (influx or efflux) or level of metalons complexing compounds, so far is not sufficient for the pur-ose of phytoremediation (with dramatically improved uptake,oot-to-shoot translocation and shoot storage) and, alternativelyor the production of healthy food (lacking non-essential elements,mproved content of nutrient elements, especially in crops grownn poor soils). Single-gene transformation in most cases does notreak significantly enough the homeostatic “barrier” at the level ofhe whole plant. Therefore, a new, more sophisticated approach isesirable, involving tissue-specific promoters inducible under cho-en conditions, and probably a multi-gene approach that wouldnable the modification of several processes governing plant metalolerance and accumulation. In addition, knowing the molecularackground of the substrate specificity would make it possible toenerate and use for the transformation transporter’s mutants withery high selectivity towards a chosen metal.

.2. Organic pollutants

Both, plant roots and leaves have been described to possesslaborate detoxification mechanisms for organic xenobiotics, firstharacterized for herbicides. It has been demonstrated that herbi-ide tolerance in numerous crops as well as resistance in weeds isaused by the action of these enzyme systems. Besides agrochem-cals, many other foreign compounds have been investigated; forxample the detoxification mechanisms have been explored withalogenated organic compounds like chloroanilines and chloroben-enes.

It has generally been accepted that several enzyme systems, notecessarily physiologically connected, form a metabolic cascade forhe detoxification, breakdown and final storage of organic xenobi-tics. Sandermann (1994) has compared this network of reactionsith a “green liver”. This cascade can be subdivided into three

istinct phases (Coleman et al., 1997) i.e. (I) activation of the xeno-iotic, (II) detoxification and (III) excretion, in analogy to human
epatic metabolism (Fig. 2). The cascade comprises of activationeactions catalysed by esterases, P-450 monooxygenases and per-xidases, true detoxification reactions in phase II performed bylutathione and glucosyl transferases, rendering the compoundnder consideration less toxic due to conjugation, and a set of

further reactions that include cleavage, rearrangement, secondaryconjugation and the like (Izryk and Fuerst, 1997; Sandermann etal., 1997; Schröder, 1997). Recently this last phase has been pro-posed to be subdivided into two independent phases, one confinedto transport and storage in the vacuole, and a second one tak-ing final reactions, e.g. cell wall binding or excretion, into account(Theodolou, 2000).

In the past, xenobiotic conjugation in plants has been investi-gated in depth for pesticides, and several isoforms of glutathioneS-transferases, glucosyl-transferases and malonyl-transferases andan array of processing enzymes have been identified in crops(Lamoureux and Rusness, 1989; Schröder et al., 2001; Schröder andCollins, 2002).

Metabolic activation of xenobiotics is in most cases cat-alyzed by P-450 monooxygenases or peroxidases (POX). Theseenzymes are localized in membrane fractions of plant cells (P-450mono-oxygenases), in the apoplast and in the cytosol (POX) (Werck-Reichhart et al., 2000; Morant et al., 2003; Passardi et al., 2005).The initial phase of chemical activation is followed by the conjuga-tion reactions as such: sugars, amino acids or glutathione may betransferred to the activated xenobiotic depending on structure ofthe molecule and its active sites. Hydroxyl, NH2-, SH- and COOH-functions on a molecule usually trigger glycosyl-transfer (Fig. 2)mediated by glycosyltransferases (GT, E.C. 2.4.1.x) (Frear, 1976;Brazier et al., 2002), whereas the presence of conjugated doublebonds or halogen-functions fosters glutathione conjugation (cat-alyzed by glutathione S-transferases, GST, E.C.2.5.1.18) (Coleman etal., 1997; Edwards et al., 2000).

Schröder and Collins (2002) have pointed out that it is crucialto know in the context of phytoremediation, which type of primaryconjugation occurs, because this will determine the final fate of thecompound (Frear, 1976).

The action of electrophilic xenobiotics in living tissue seemsto depend on their nucleophilic cellular counterparts. There is apreference for the reactions between xenobiotics and biomolecu-lar partners which may be explained by the concept of hard andsoft nucleophiles/electrophiles (Coleman et al., 1997). Any reac-tion with hard electrophiles requires additional enzymatic support,which may be provided by glutathione S-transferase isoenzymes.In any case, detoxification totally depends on the availability ofglutathione. The homeostasis of glutathione inside the plant ismaintained by a complex regulation process including synthesis,degradation and long range transport (Noctor et al., 2002).

Numerous herbicides are conjugated to sugars via O- orN-glucosyl-transfer. These reactions are catalyzed by differentenzyme families (Messner et al., 2003). It is significant that con-jugation may occur directly, but that sometimes activation maybe needed in advance to provide the xenobiotic with the respec-tive activated sites (see above). As P-450 monooxygenases act onmany of the mentioned compounds, hydroxylation will favourO-glucoside formation. For N-glucosyl transfer, coupling to NH2-groups of the molecule is crucial. In summary, goals for themetabolic control of any remediation process will have to take intoaccount activating or detoxifying enzymes, but also the availabil-ity of the conjugation partners, e.g. UDP-glucose, amino acids, orglutathione and its analogues.

Attempts to increase the detoxification potential of plantstowards organic pollutants have mostly been studied with regardsto pesticides but, unfortunately, not with other pollutants. But evenin the case of pesticide tolerance, the available GMO plants containmicrobial genes that cleave the active substance, rather than over-
expressing plant genes. Of course, it is technically feasible to intro-duce bacterial genes for the cleavage of PAH, benzenes, and evenPCB into plants, but the market for such an approach is inexistent.
Despite this, the potential to increase detoxification capacitiesfor organic xenobiotics exists. Numerous studies have demon-

18 J.A.C. Verkleij et al. / Environmental and Experimental Botany 67 (2009) 10–22

Fig. 2. Metabolic cascade for the detoxification, breakdown and final storage of organic xenobiotics (modified after Schröder and Collins, 2002). After uptake into the cell,xenobiotics with electrophilic functions (here: a classical diphenyl ether herbicide) will be attacked by Phase II enzymes, like the glutathione S-transferases or glycosyl-transferases (GT). Conjugates (R-SG) are frequently sequestered in the vacuole by tonoplast ATPases (Theodoulou, 2000; Schröder et al., 2001; Grzam et al., 2007), but novelevidence points at the existence of alternative pathways leaving the conjugate in the cytosol or translocating it into the apoplast (Ohkama-Ohtsu et al., 2007). Vacuolarcleavage of GS-conjugates may occur by a sequence of carboxypeptidase (CP) and dipeptidase (DP) reactions (Wolf et al., 1996), or by g-glutamyltranspeptidase (Grzam eta detaia 1991;( ith timt

sct

idasx

otptvoarth

TP(

SSSSSSS

R

l., 2007). The resulting cysteinylconjugates (R-SCys) have not been investigated intoxification of such conjugates by intestinal bacteria has been observed (Anders,

Dekant, 2003). However it is assumed that normal plant metabolism will proceed werminal metabolites with the cell wall.

trated that herbicide safeners will induce the detoxificationascade, and also ABC-transporters promoting conjugate seques-ration in the plant vacuole.

Besides, light oxidative stress, for example by UV-radiation, elic-tors or even by heavy metals, will induce the activity of certainetoxification enzymes, predominantly P-450 monooxygenasesnd glutathione S-transferases. For herbicide safeners, a putativeequence of steps necessary to induce plant tolerance towardsenobiotics has been discussed (Table 1).

Even more important, when the dualities of heavy metal andrganic pollutant occurrence are taken into account, any stimula-ion of glutathione biosynthesis, perhaps also by heavy metals, willrobably also be beneficial for conjugation reactions and seques-ration of foreign compounds. This is specifically important withiew to novel results indicating a long range transport and export
f glutathione conjugates in plant roots (Schröder et al., 2007). Suchn excretion of potentially dangerous compounds from the plant,esulting in lower pollution content, seems possible under condi-ions of high availability of sulphur, and glutathione, and wouldence be stimulated if GSH biosynthesis was stimulated.
able 1roposal for a sequence of stages that lead to safener induced xenobiotic tolerancefrom Izryk and Fuerst, 1997, modified after Schröder, 1997).

tage 1: Uptake of safener by seedlings into root and/or shoottage 2: Metabolism of safener to active form by phase I enzymestage 3: Specific signal recognition and signal transductiontage 4: Increased transcription of GSH synthesis and GST genestage 5: Increased levels of GSH and GST isoformstage 6: Increased xenobiotic conjugationtage 7: Sequestration of conjugates in vacuole

esult: Enhanced whole-plant tolerance to organic pollutants

l. However, in cattle fed with plant material containing xenobiotic GS-metabolites,Sandermann et al., 1992), similar to rats fed with haloalkane containing materialse towards S-glucosides, sulphonates or related compounds, and finally yield bound

5. Dualities in plant tolerance to pollutants: consequencesand solutions

We have taken a comprehensive approach to review plant tol-erance to pollutants. There is an inherent duality in plant toleranceto heavy metals. On one hand tolerant plants having heavy metaltolerance and hyperaccumulation potential could be beneficial inphytoremediation for clean-up of soil and water; on the other handtolerant food crops, if exposed to heavy metals in their growthmedium, may be dangerous as carriers of toxic metals or metabo-lites of organic pollutants in the food chain leading to food toxicity.Still another aspect of this duality lies in the potential use of tol-erant crops for fortification of food with essential metals (Guerinotand Salt, 2001) or with the enhanced detoxification of organic com-pounds. Using the food/spice plant Allium schoenoprasum (Chives)as a model plant for study of this duality it has been shown thatchives is highly tolerant to Cd and nickel and to a lesser extent tocobalt (Golan-Goldhirsh, 2006). The tolerance of chives to heavymetals is also reflected in its ability to accumulate Fe. In addi-tion to being a food crop tolerant to heavy metals chives is richin sulphur compounds. Some of them, such as cysteine and glu-tathione can serve as reductants or as scavengers of metals toprevent oxidative damage. An increase in total SH-containing com-pounds in the root of chives under Cd stress conditions has beenreported (Barazani et al., 2004). This is accompanied by a reduc-tion in glutathione concentration in root, which raises the questionabout the identity of the newly synthesized SH compounds induced
under the Cd treatment. Chives is a better accumulator of Cd thanmost other food crops reported in the literature. It accumulatesmore than 0.1% of shoot dry weight in Cd, which seems to cat-egorize it as a Cd hyperaccumulator (Baker and Brooks, 1989).Furthermore, chives shoots can be re-harvested many times during

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growing season by that multiplying the amount of metal removedrom the medium. It takes 6–9 weeks before anatomical changesnd standard physiological and biochemical indicators (biomassccumulation, photosynthetic activity, chlorophyll and carotenoidsoncentration, etc.) are detected. However, detoxifying enzymesGSTs) are induced within 24 h of metal addition to the medium andhen levels off with control. Thus, chives appear to be an ideal planto follow metabolic adjustments to maintain homeostasis undertress.

Little is known about the impact of heavy metal ions on theetabolic profile and metabolic flux in plants. It has recently been

hown using proton NMR spectroscopy of chives treated with Cdhat several aliphatic amino acids including asparagine, aspartate,lutamine, glutamate, proline, serine and threonine are the majormino acids in the extracts that vary in concentration in differentlant parts under stress (Ostrozhenkova, 2008).

The other side of the coin of plant tolerance to heavy met-ls and phytoremediation is fortification of tolerant food cropsith essential metals. Iron deficiency is considered to be the most

ommon nutritional deficiency that affects approximately 20% ofhe world population. Women and children are especially at riskhttp://www.who.int/nut/research1.htm). Promoting iron-fortifiedlant foods is necessary to reduce iron deficiency, as most of theeople of the world get their iron from eating plant products. Ironeficient humans and animals are more vulnerable to stress andarasite infection. In preliminary experiments it has been foundhat chives accumulated up to approx. 0.5 mg Fe per gram dryeight. This sets chives among the rich sources of plant Fe. Just

or comparison, Fe content in a few major food crops, on a mg g−1

asis: beans, 0.055; rice, 0.022; wheat, 0.053; corn 0.020. The poten-ial advantage of chives is obvious. Nevertheless, it should be saidt the outset that we do not suggest that chives can solve the ironalnutrition problem in the world, because it is not a staple foodhere it is needed mostly, and the daily consumption even in pop-

lations that consume fresh chives regularly won’t make up for theaily requirement. However, we contend that iron- fortified driedowder of chives could be a useful additive for food/feed products.ost importantly it serves here to make the point of the duality in

rop plant tolerance to heavy metals.Environmental pollution by trace elements and especially by

eavy metals is in many cases accompanied by pollution withrganic foreign compounds and vice versa. Phytoremediation butlso phytostabilisation in such areas affected by multiple pollu-ion is complicated, and only few plant species have been shown tourvive under such adverse conditions.

One of the main problems for any given plant species/cultivars its survival under mixed pollution situation where heavy met-ls might occur together with organic pollutants, as in the casef military training sites or many industrial brownfields, whichas not been investigated in any depth (Chaudhry et al., 2001).

t is obvious, that quite a number of remediation projects haveailed due to insufficient plant performance. Recent studies havehown that Typha and Phragmites, but also mutants of N. tabacumLyubenova et al., in press) are capable of removal and metabolismf heavy metals and organic pollutants to some extent, but will failt higher concentrations. Pollution with heavy metals will interfereith both, the oxidative stress defence in plants, and with their abil-

ty to conjugate organic xenobiotics (Schröder et al., 2002). Despitelant species dependent differences, the general reactions seem to

nclude oxidative stress and an induction of anti-oxidative enzymes.everal processes seem to depend on direct binding of heavy metals
o enzyme proteins, but effects on transcription are also observed.nduction of xenobiotic metabolism will be obtained at high heavy
etal concentrations, when plant stress is elevated.Trace elements are well known for their ability to interfere with

he plant’s metabolism and to induce the formation of reactive


oxygen species (ROS), albeit through different reactions. On theother hand, it has been shown that ROS may serve as signallingmolecules for a number of defence reactions in plants, includingalterations in the sulphur metabolism. Tightly connected to thesulphur metabolism in plants is the predominant detoxificationpathway of halogenated organic pollutants and herbicides, i.e. glu-tathione dependent detoxification. Here, it might be speculated thatconsumption of GSH for heavy metal sequestration and for con-jugation of xenobiotics could also lead to additional plant stress.Under real life conditions and multiple pollution scenarios thismight mean that a combination of heavy metals and organic pollu-tants (a) can be tackled by the plant, if the correct enzyme activityis induced, or (b) leads to rapid development of stronger stress dueto the additional action of the un-detoxified organic xenobiotics.

Acknowledgments

This manuscript is the outcome of a collaboration betweenmembers of Working Group 1 of COST Action 859: Plant uptake/exclusion and translocation of nutrients and contaminants. Allauthors have joined and still participate in this important COSTAction concerning: “Phytotechnologies to promote sustainable landuse and improve food safety”.

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