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V. Options to Reduce the Cadmium Content in Tobacco Leaves

V. Options to Reduce the Cadmium Content in Tobacco Leaves

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percentage reduction of Cd in tobacco leaves than the other approaches. Research

in many of these areas is still at relatively early stages and the available information

is often limited to studies using other plants. Based on our current knowledge, it is

reasonable to expect that most of the metal transport and sequestration

mechanisms observed in other plant species are relevant for tobacco as well,

and therefore have been included in the discussions in this section.



1.



Root Exudates



Root exudates containing, for example, low-molecular-weight organic acids

(LMWOA), may induce changes in the physicochemical characteristics of the

surrounding soil, such as pH, moisture, electrical conductivity, redox potential,

oxygen availability, or microbial community (Hinsinger et al., 2003; Jones et al.,

2003). Hence, they may affect the solubility of various soil components (e.g., Cd)

and thus the availability of such components to plant roots. However root

exudates do not necessarily explain differences in Cd accumulation between taxa

(Zhao et al., 2001). In the rhizosphere, organo-Cd complexes may account for a

significant portion of the soil solution Cd (Jones et al., 1994). In particular, citrate

can efficiently solubilize Cd (Naidu and Harter, 1998; Nigam et al., 2002) and its

exudation may enhance Cd solubility in the rhizosphere. As LMWOA may play a

role in Cd solubilization and accumulation in plants (Cieslinski et al., 1998),

genes that facilitate their release could be introduced by genetic engineering to

reduce or enhance Cd uptake (Ryan et al., 2003). The concept of phytoextraction

is further discussed in Section V.C.1.

Because root cells are mostly mature cells with large vacuoles, vacuolar

chelation may predominate over cytosol mechanisms (Rauser, 1999). Wagner

(1995) argued that, at the low levels of Cd found in agricultural soils, little or no

PCs would be induced, and vacuolar citrate would effectively complex

cellular Cd.

In response to nutrient metal ion deficiencies, such as Fe, graminaceous plants

secrete phytosiderophores (e.g., mugenic and avenic acids) to increase

the bioavailability of soil metals and help to carry the metals into plant tissue

(also see Section V.A.2(a), for a discussion of Cd transport under Fe-deficient

conditions). For example, mugenic acids may limit the binding of Cd by hydrous

Fe-oxide (Mench et al., 1994b). Phytosiderophores can mobilize Cd from a solid

phase even in the presence of the competing metals, Fe, Ca, and Mg, but their

presence did not result in a significant increase in Cd uptake by barley and wheat

(Shenker et al., 2001). This suggests that the release of phytosiderophores may

not increase Cd phytoextraction efficiency (see Section V.C.1 for a discussion of

phytoremediation). In contrast, phytosiderophores may be able to reduce Cd

uptake. When maize was exposed to Cd, in hydroponic culture, in the presence of



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root exudates containing 20 -deoxymugineic acid, a prime maize phytosiderophore, the plants accumulated less Cd than did the corresponding experimental

controls (Hill et al., 2002). While this result may be due to increased production

of phytosiderophores under Fe-limiting conditions, other root exudates may have

played a role. These results need to be verified in conditions that better mimic the

field situation.

The results of these studies suggest that organic acids that are present in

the rhizosphere soil may play a role in the solubilization of particulate-bound

Cd into soil solution and its subsequent accumulation in plants. However, it

should be emphasized that the responses will depend on various factors (e.g.,

soil characteristics, plant species). Moreover, the sorption of organic acids to

the mineral phase and the mineralization by soil microorganisms play a key

role in determining the effectiveness of organic acids in most rhizosphere

processes (Jones, 1998). Soluble root exudates of N. tabacum (cv. PBD6)

extract more Cd than those of N. rustica (cv. Brasilia) and much more than

exudates of Zea mays (Mench and Martin, 1991). The extent of Cd extraction

by root exudates correlated with Cd bioavailability to these three plants when

grown in soil. An increase in Cd solubility in the rhizosphere of apical root

zones due to root exudates is likely to be an important cause of the relatively

high Cd accumulation in Nicotiana spp. (Mench and Martin, 1991). Although

the nature of these exudates was not identified, it is possible that organic

acids were the major components responsible for the increased Cd, Mn, and

Cu extraction by Nicotiana root exudates. Krotz et al. (1989) examined the

possible involvement of vacuolar organic acids in the accumulation of Cd

and Zn in cultured tobacco cells exposed to non-growth-inhibiting and

growth-inhibiting levels of these metals and in the presence and absence of

Cd-peptide (phytochelatin). They concluded that tobacco suspension cells can

accumulate Cd and Zn in the form of vacuolar organic acid (mostly malate

and citrate) metal complexes.

To our knowledge no transgenic tobacco has been engineered to express a

protein involved in the biosynthesis of organic acids, with the aim of reducing Cd

levels in the leaves. Such manipulation might impact respiratory control by

disrupting malate and citrate homeostasis.



2.



Cadmium Transporters



Nutrients needed by the plant are often present in the soil solution in low

amounts (e.g., P, N). Therefore, plants must use high-affinity transport systems to

accumulate these ionic nutrients. Cadmium, as well as other non-essential trace

metal(loid)s such as Cr, Hg, and arsenic (As), are most likely carried across plant

membranes via transporters, which may represent the means by which to modify



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Cd transport. Although various enzymes are known to transport Cd (Guerinot,

2000; Theodoulou, 2000; Williams et al., 2000; Clemens, 2001), no Cd-specific

transporters have been unambiguously identified to date. Indeed, few ion

transporters are shown to be absolutely ion specific. In addition, the regulation of

the genes encoding these transporters may be complex, with regard to

transcriptional and post-translational regulation.

A few transporters that may participate in Cd uptake, accumulation, and

mobilization are discussed below and are presented in Fig. 5. Their sub-cellular

location(s) have generally not been established and an understanding of the

regulation of all transporters is in its infancy. Also, additional transporter types

will undoubtedly be identified before long (Maăser et al., 2001). For example,

Gupta et al. (2002) recently defined a new class of heavy-metal (e.g., Cd)

binding, histidine-rich proteins called metallohistins.

(a) ZIP family. The ZIP family of metal transporters takes its name from zinc

regulated transporter- (Z RT) and iron regulated transporter- (I RT) like Proteins

(Grotz et al., 1998) (reviewed in Guerinot, 2000; Maăser et al., 2001). The yeast



Figure 5 Possible transport mechanisms for Cd uptake and accumulation in plant cells (the cell

wall is not shown), including one non-plant transporter (denoted by p) that may be used for genetic

engineering. Experimental evidence for most transporters depicted is limited, with the exception of

tonoplast CAX and the yeast YCF1 transporting Cd –GSH. Similarly, sub-cellular localization of

many of the possible mechanisms shown has not been established. Note that the YCF1 protein was

tentatively located in the transgenic plant tonoplast. The relative importance of various mechanisms

may depend on Cd exposure level, tissue, developmental stage, species, and other factors. Increasing

evidence suggests that the regulation of transporters is complex and integrative. An understanding of

which mechanisms primarily function in a particular plant and growth condition will undoubtedly be

advanced by integrative studies using transcriptomics and proteomics.



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ZRT1 gene encodes a high affinity transport system for Zn, which can also

transport Cd (Guerinot, 2000; Gomes et al., 2002).

Fifteen members of the ZIP family have been identified in Arabidopsis (Maăser

et al., 2001), some of which can transport Cd across the plasmalemma. In plant

roots, the IRT1 gene is transcriptionally responsive to Fe deficiency, but once

expressed it may be capable of transporting Cd, for example, in addition to Fe

(Eide et al., 1996; Cohen et al., 1998), as occurs in yeast expressing A. thaliana

IRT1 (Korshunova et al., 1999). Pea seedlings grown in Fe-deficient hydroponic

conditions with 0.2 mM Cd(NO3)2 for 2 days contained approximately twice the

amount of Cd in the roots, but about three times less Cd in the shoot, compared to

seedlings grown in Fe-sufficient conditions (Cohen et al., 1998). Interestingly,

under Fe-deficient conditions, IRT1 appeared to be responsible for a significant

increase in Cd uptake by the ecotype Ganges of Thlapsi caerulescens, but not in

the Prayon ecotype, which is less efficient at hyperaccumulating Cd (Lombi et al.,

2002). Regulation of IRT1 is both at the transcript and protein levels. Under Felimiting conditions, overexpression of this gene led to constitutive expression of

the mRNA, but the protein was present only in the roots (Connolly et al., 2002).

IRT1-transgenic Arabidopsis accumulated more Cd than wild-type plants

(Connolly et al., 2002). Cadmium specificity of the IRT1 transporter may be

enhanced by genetic engineering, as suggested by results obtained in studies of

yeast by Rogers et al. (2000). They substituted amino acid residues in IRT1 that

are conserved among ZIP family members and created yeast mutants limited in

the ability to transport Cd. While this transporter may be an interesting means by

which to modify Cd uptake by plants, little is currently known regarding its

properties, e.g., mechanisms of regulation.

While A. thaliana ZIP1, ZIP2, and ZIP3 (and soybean GmZIP1; Moreau et al.,

2002) may all transport Cd to some extent, the inhibition of Zn uptake in yeast by

Cd was the most severe for ZIP2 (Grotz et al., 1998). Other members of this

family may transport Cd; T. caerulescens TcZNT1 has been shown to mediate

low-affinity Cd uptake (Pence et al., 2000). Recently, Assunc¸a˜o et al. (2001) have

cloned a homolog of ZNT1 in T. caerulescens (TcZNT2).

(b) ABC transporters. The ATP binding cassette (ABC) superfamily is a large,

ubiquitous, and diverse group of proteins, most of which mediate transport across

biological membranes. ABC transporters are MgATP-dependent, vanadateinhibited pumps that do not depend on the vacuolar proton gradient. The study of

plant ABC transporters is relatively new, but it represents a growing field of

investigation (e.g., Rea, 1999; Theodoulou, 2000; Martinoia et al., 2002).

Full-size ABC transporters have been best characterized, to date. The major

groups of these full-size proteins are the multidrug-resistance proteins (MDR), or

P-glycoproteins (PGP), the multidrug-resistance-related proteins (MRP)

and protein products of the pleiotropic drug resistance genes (PDR)

(Martinoia et al., 2002).



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In yeasts and plants, some ABC transporters were shown to confer Cd

tolerance. HMT1 is a Schizosaccharomyces pombe gene that encodes a half-size

ABC protein that transports apo-PCs and PC –Cd complexes across the tonoplast

(Ortiz et al., 1995). There are apparently no authentic HMT1 homologues in

Arabidopsis, implying that plants presumably use other ABC transporters for the

vacuolar uptake of Cd (Sanchez-Fernandez et al., 2001). The yeast Cd factor

(YCF1) belongs to the MRP family and can mediate Cd resistance (Szczypka

et al., 1994; Wemmie et al., 1994; Tommasini et al., 1996). The YCF1 gene,

regulated by the yAP1 transcription factor (Wemmie et al., 1994), encodes a

vacuolar pump capable of transporting organic glutathione (GSH) conjugates and

Cd – GSH complexes. The protein catalyzes the uptake and hence, the vacuolar

sequestration, of the Cd – GSH complex, Cd2·GSH2 [bis(glutathionato)-Cd]

(Li et al., 1997). The structure of Cd2·GSH2 is close to that of Cd2·PC2 (a PC –

Cd complex). However, the latter is not transported by the YCF1 protein (YCF1p)

(Li et al., 1997), although plant PCs are known to chelate Cd (see Section V.A.3).

Interestingly, yeasts bearing the change Trp1225 to Cys in a transmembrane

domain of the YCF1p, tolerated a Cd concentration ninefold higher than the wildtype cells (Falco´n-Pe´rez et al., 2001). YCF1-deficient yeast cells (Dycf1) removed

9% of the initial Cd from the growth medium after 24 h, compared with 23% for

the control strain (Gomes et al., 2002). The hydrophobic N-terminal extension

(a characteristic found in many MRP proteins) of YCF1p contains a cytosolic

linker region essential for Cd resistance (Mason and Michaelis, 2002). Tobacco

(cv. SR1) has been transformed with the YCF1 gene, as well as with the PDR5

gene, but transcripts of both genes were shorter than expected (Grec et al., 2000).

Recently however, this gene was successfully overexpressed in Arabidopsis

(Song et al., 2003). Transgenics had increased tolerance to, and accumulated

more, Cd and Pb, suggesting that this gene may be useful for Cd phytoextraction

(see Section V.C.1). The yeast Bpt1p, an YCF1p homologue, appears to play a

minor role in Cd transport (Klein et al., 2002; Sharma et al., 2002).

A plant member of the MRP family, AtMRP3, is able to complement yeast

YCF1-deletion mutants that are sensitive to Cd (Tommasini et al., 1998). In

Arabidopsis, transcript levels of this gene are increased both in roots and shoots

of 7-day-old plantlets exposed to Cd. In 4-week-old plants, it is only upregulated

in the roots (Bovet et al., 2003). Therefore, the developmental stage appears to

play an important role in the expression of this gene in this species.

(c) Nramp family. The natural resistance-associated macrophage protein

(Nramp) family defines a family of related proteins that are likely implicated in

the transport of divalent metal cations across the plasmalemma. Homologues

have been found in a wide range of living organisms, including higher plants

(Williams et al., 2000).

A. thaliana Nramp 1, 3, and 4 were able to complement yeast mutants

defective in Mn and Fe uptake (Thomine et al., 2000; Williams et al., 2000).

AtNramp1 mRNAs are preferentially expressed in Arabidopsis roots, whereas



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AtNramp3 and 4 are expressed in roots and aerial parts (Thomine et al., 2000).

Interestingly, AtNramp1, 3, and 4 expression in yeast leads to an increased Cd

sensitivity and Cd accumulation (Thomine et al., 2000). Arabidopsis seedlings

grown on metal-replete medium and overexpressing AtNramp3 displayed Cd

hypersensitivity, as assayed by root growth measurements (Thomine et al.,

2000). Disruption of AtNramp3 in Arabidopsis (T-DNA tagged line) led to a

moderate increase in Cd resistance (increased root growth compared with the

control, in medium with increased Cd concentration), but there was no significant

difference in Fe, Mn, and Zn content or Cd accumulation levels in the plants

(Thomine et al., 2000).

The Saccharomyces cerevisiae SMF1 and SMF2 genes encode membranetransport proteins that are able to transport Mn, as well as other metals, including

Cd (Liu et al., 1997). In yeast, Cd uptake by the SMF1 protein is downregulated

by the BSD2 protein, preventing the accumulation of Cd in the cell. It was

shown that inactivating the BSD2 gene resulted in bsd2 mutant cells that

accumulated high levels of Cd, because SMF1 is stabilized, instead of entering

the vacuole where it is degraded (Liu et al., 1997; Liu and Cizewski Culotta,

1999). Metals also play an important role in the post-translational regulation of

the SMF1 protein.

(d) P-type ATPase. P-type ATPases have been identified in a wide range of

organisms as diverse as bacteria, yeasts, and humans. This large superfamily uses

ATP to energize the transport of a variety of ions across biological membranes.

These P-type ATPases are distinguished in their formation of a phosphorylated

intermediate (hence, called P-type) during the reaction cycle (Williams et al.,

2000; Axelsen and Palmgren, 2001).

Several bacterial ATPases are involved in Cd transport, like Escherichia coli

ZntA catalyzing Cd, Zn, and Pb efflux (e.g., Silver, 1996; Lee et al., 2001). In

Arabidopsis, the P1B ATPase subfamily contains eight metal transporting

members, four of which (the proteins are named HMA1-4) are thought to be

involved in Zn/Co/Cd/Pb transport across the plasmalemma (Mills et al., 2003).

They are also called CPx-ATPase because they share the common feature of a

conserved intramembrane cysteine-proline and either a cysteine (Cys), histidine

(His), or serine motif (CPx motif), which is thought to function in metal transport.

The type of metal-binding motif, as well as the type of residues close to the

motifs, may be involved in the metal selectivity or affinity for a particular metal at

the binding site (Williams et al., 2000). Yeasts expressing AtHMA4 are more

resistant to Cd. In Arabidopsis, AtHMA4 is expressed at the highest levels in roots

and is downregulated in roots after exposure to 1 mM CdSO4 for 30 h (Mills et al.,

2003). Some P1B P-type ATPases may have potential as Cd transporters, but their

exact role in plants remains to be elucidated.

(e) CDF family. Members of the cation diffusion facilitator (CDF) protein

group of metal transporters that are involved in the transport of Zn, Co, and Cd

have been identified in bacteria, fungi, plants, and animals. Eukaryotic members



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of the CDF family (or cation efux family; Maăser et al., 2001) share a His-rich

region, which may be involved in metal binding. Certain members of the CDF

family are thought to function in metal efflux or vacuolar uptake; some are found

in plasma membranes, while others are in intracellular membranes (reviews in

Williams et al., 2000; Maăser et al., 2001). For example, the yeast ZRCl is

involved in Cd tolerance, probably by transporting Cd into the vacuole (Li and

Kaplan, 1998; Maăser et al., 2001). In plants, the Zn transporter of A. thaliana

(AtZAT1) was the first CDF member to be characterized (Van der Zaal et al.,

1999). When overexpressed in transgenic Arabidopsis, a slight, but significant

increase in Zn tolerance is observed at high Zn exposure, and Zn accumulates in

the roots, but not in the shoots (Van der Zaal et al., 1999). However, Northern

blot analysis indicates that ZAT1 is expressed throughout the plant (Williams

et al., 2000). It might be involved in vacuolar sequestration of Zn (Van der Zaal

et al., 1999; Guerinot, 2000). Antisense plants were viable, and had a wild-type

level of Zn resistance and accumulation (Van der Zaal et al., 1999). By

reconstituting the ZAT1 protein into proteoliposomes, Bloss et al. (2002) showed

that 109Cd transport rate by ZAT1 represents only 1% that of Zn. Moreover, when

incubated with Cd, the bacterium Ralstonia metallidurans expressing AtZAT1p

did not accumulate different amounts of Cd, compared with controls (Bloss et al.,

2002). Other ZAT-related proteins (or metal tolerance protein, MTP) are found in

Arabidopsis and other plants. MTPs from Thlaspi goesingense may play a role in

the vacuolar sequestration of Cd (Maăser et al., 2001; Persans et al., 2001). More

studies are needed on CDF proteins to determine their functions, expression

patterns, and possible role in Cd transport.

(f) Cation/proton antiporters. Antiporter proteins can exchange protons (Hỵ)

for metal ions in the vacuole sap, causing the accumulation of the metals in the

vacuole. Such a transport was shown for Cd across the vacuole membrane of oat

roots (Salt and Wagner, 1993; Gries and Wagner, 1998; Gonzalez et al., 1999). It

has also been shown for tobacco roots vesicles (Koren’kov et al., 2002).

However, it is not clear whether this mechanism is a lower affinity metal

transport, analogous to that which is associated with the Ca2ỵ/Hỵ antiporter

(Williams et al., 2000). Further investigation is needed to determine whether

distinct proteins transport Ca and Cd (Koren’kov et al., 2002). It has been

suggested that the CAX1 and CAX2 (calcium exchanger 1 and 2) genes, which

encode putative divalent metal/Hỵ antiporters from Arabidopsis (Hirschi et al.,

1996), may also transport Cd (see below and Fox and Guerinot, 1998). Hirschi

et al. (1996) suggested that CAX2, which is located in the vacuolar membrane in

Arabidopsis, may be involved in metal (e.g., Cd) transport by plants. Yeast

vacuolar Ca transport was increased when nine amino acids of the CAX1 Ca

domain were inserted into CAX2 (CAX2 –9; Shigaki and Hirschi, 2000; Shigaki

et al., 2001). Tobacco (cv. KY160) expressing CAX2 under control of the 35S

promoter accumulated three times more Cd in the roots and 15% more Cd in

stems than controls. It also accumulated more Ca and Mn, suggesting that CAX2



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has broad substrate selectivity. Modification of this transporter may be an

important component of future strategies to improve plant divalent ion tolerance

(Hirschi et al., 2000). CAX4, cloned from an Arabidopsis cDNA library, is

expressed in all tissues analyzed, although seemingly at levels lower than CAX2

(Cheng et al., 2002). CAX4 RNA levels increased slightly, in response to Mn, Ni,

and Na. CAX4 suppressed, although weakly, the Cd sensitivity of a yeast

IRT1-expressing strain (IRT1 transports Cd into the cytosol and thereby is

assumed to make the wild-type yeast strains more Cd sensitive) (Cheng et al.,

2002). This suggests that CAX4 may transport Cd. In tobacco, CAX4 is located in

the vacuolar membrane (Cheng et al., 2002). Alterations at the N-terminus of the

CAX transporter genes may modulate their ion transport properties.

(g) LCT1. The wheat low affinity cation transporter (LCT1) cDNA (Schachtman et al., 1997) was found to increase the uptake of Ca and Cd when expressed

in yeast cells (Clemens et al., 1998). A mild pH dependence in Cd uptake in

transgenic yeast was noticed, with uptake rates highest at pH 6. Calcium

interfered strongly with the uptake of Cd. It has been proposed that in plants,

LCT1 functions as a Ca transport system and might contribute to Cd transport,

except in soils with high Ca levels (Clemens et al., 1998). Recently, Amtmann

et al. (2001) expressed wheat LCT1 in a salt-sensitive yeast mutant lacking a Na

export pump. The transformed yeast showed enhanced Na accumulation and loss

of intracellular K (were NaCl sensitive). However, high K and Ca concentrations

in the growth medium inhibited Na uptake through LCT1 and hence, rescued the

growth of the LCT1-transformed yeast mutant on saline medium. LCT1 cellular

localization in plants is unknown (plasma or internal membrane).

(h) MATE family. The multidrug and toxic compound extrusion (MATE)

family has been recently defined (Brown et al., 1999). Li et al. (2002) isolated a

MATE efflux protein from Arabidopsis, At DTX1 (for detoxification 1), which is

probably located in the plasma membrane. KAM3 mutant bacterial cells are Cd

sensitive. They did not grow on the medium containing 10 mM Cd or higher, but

when transformed with AtDTX1, they grew in the presence of up to 100 mM Cd.

This suggests that AtDTX1 may function as an efflux carrier to extrude toxic

compounds in plant cells. Such systems are well established in bacteria. Based on

their results, Li et al. (2002) speculated that AtDTX1 could be required to export

the ions into the xylem for long-distance transport. This study shows that plant

cells possess at least one efflux mechanism, in addition to internal sequestration

mechanisms, for metal detoxification. But, it remains to be seen if efflux systems

are restricted to long-distance transport tissues.

(i) Cation channels. While relatively little is known about Cd passage through

the channels of plant membranes, available published evidence suggests that

plasmalemma Ca channels may play a prominent role in the uptake of Cd into

the cytosol. Cadmium has been considered a channel blocker in animal systems,

but a number of studies have demonstrated its permeability through Ca channels

(see Perfus-Barbeoch et al., 2002). Calcium is established as a signal transduction



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messenger and amplifier in stomatal function (White, 2000). Recently,

Perfus-Barbeoch et al. (2002) concluded that Cd affects guard cell regulation

in an ABA-independent manner after its entry into the cytosol through Ca

channels, but not K channels, perhaps by perturbing Ca-calmodulin or Ca-ionmediated signaling processes. The effects of low Cd treatment (10 pM) are

perhaps particularly interesting because they suggest that if root cell Ca channels

are similarly sensitive to Cd, channel-mediated permeation may be a significant

route for entry of Cd into roots under low-level Cd exposure, as occurs in the

typical field situation. It is apparently not known if tonoplast channels

(e.g., SV channels) may participate in Cd efflux from root cells during Cd

translocation to the shoot. These channels are thought to be permeable to various

monovalent and divalent cations (White, 2000).



3.



Phytochelatins, Metallothioneins, and Glutathione



Phytochelatins (PCs), metallothioneins (MTs), and glutathione (GSH) are

cysteine-rich, low-molecular-weight polypeptides that can bind various metals.

The role of plant PCs and MTs in metal tolerance and detoxification have been

the subject of various recent reviews (e.g., Cobbett and Goldsbrough, 2002).

(a) Metallothioneins in plants. In mammalian systems, MTs can bind to

metals such as Cd, to protect the cell from toxicity. Animal and yeast MTs are

small (approximately 60 amino acids, of which 20 are Cys) gene-encoded

proteins consisting of two domains and capable of binding a total of seven

divalent metal ions. In their reduced state they provide thiols for metal chelation

through mercaptide bonds. The arrangement of Cys residues in different MTs

may affect the metal-binding specificity. Metallothionein proteins are difficult to

isolate because of their high sensitivity to oxygen of the thiol groups. The first

evidence of MTs in plants (wheat) was provided by Lefebvre et al. (1987), and

the first MT-like gene isolated and sequenced in monocotyledon plants was

apparently rgMT-1, a rice stress-inducible MT-like gene (Hseih and Huang,

1998). A number of MT-like proteins have been cloned from several plant

species and appear to be expressed at relatively high levels (Zhou and

Goldsbrough Peter, 1994; Choi et al., 1996; Liu et al., 2002). However, there is

no evidence that they function in metal scavenging in plants (Rauser, 1995;

Zenk, 1996), and the level of knowledge about MTs is poor, compared with the

insight gained about PCs. Nonetheless, the list of MT-like genes found in plants

has grown to 58 from a range of plants (including tomato) and tissues (reviewed

in Rauser, 1999). Recently, it was found that banana MT3 gene expression was

greatly enhanced in response to CdSO4, copper sulfate (CuSO4), and zinc

sulfate (ZnSO4) (Liu et al., 2002). In a brief study, Watanabe et al. (2001)

expressed yeast CUP1 gene in sunflower at the callus stage and found increased



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resistance to Cd. Hong et al. (2000) improved the stability of human MT (hMT)

protein in transgenic E. coli by designing synthetic genes for dimeric and

tetrameric hMTs. The oligomeric MT bound twice the amount of Cd as did

monomeric hMT.

(b) Phytochelatins. The term phytochelatin (PC) was proposed by Grill (1987)

to designate a class of peptides induced by metal exposure in plants.

Phytochelatin biosynthesis occurs within minutes of Cd exposure. The general

structure of PCs is (g-Glu-Cys)n-Gly n ẳ 2 11ị: Phytochelatin variants can

have residues other than glycine (Gly) at the C-terminus (Zenk, 1996). According

to Zenk (1996), PC binding affinity in plant suspension cells is Cd . Pb .

Zn . Sb . Ag . Hg . As . Cu . Sn . Au . Bi.

Phytochelatins are not gene encoded, but are instead the product of a

biosynthetic pathway. They are synthesized in plants from GSH or its analogues

by the phytochelatin synthase (PCS or PC synthase), an enzyme activated by Cd

and other metal ions (Grill, 1987; Grill et al., 1989). Genes encoding PCS have

been recently identified in yeasts and plants (wheat, Arabidopsis) and

homologues were also found in the nematode Caenorhabditis elegans (Clemens

et al., 1999; Ha et al., 1999; Vatamaniuk et al., 1999).

The Arabidopsis PCS1 (AtPCS1 ¼ CAD1) gene suppresses the Cd-sensitive

phenotype in yeast (Ha et al., 1999; Vatamaniuk et al., 1999). The level of

AtPCS1 mRNA increased 2.1-fold in wild-type 5-day-old Arabidopsis seedlings

subjected to 50 mM Cd, compared with non-treated plants (Lee and Korban,

2002). AtPCS1 is transcriptionally regulated by Cd at the early stage of

development, but this regulation disappears as the plants grow older (Lee and

Korban, 2002). In 10- and 21-day-old plants, Ha et al. (1999) and Vatamaniuk

et al. (1999), respectively, did not detect transcriptional regulation of AtPCS1.

A second Arabidopsis gene, AtPCS2, when expressed in yeast, confers Cd

resistance at exposure levels up to 100 mM CdCl2, when compared to control

yeasts (Cazale´ and Clemens, 2001). In Arabidopsis, AtPCS2 is not upregulated by

exposure to 10 mM Cd and is expressed at a relatively low level, compared with

AtPCS1, in most plant tissue. However, it has been argued that it may be the

predominant PC synthase in some tissues or environmental conditions,

because this enzyme has been preserved as a functional PCS through evolution

(Cobbet, 2001).

Transgenic S. cerevisiae, expressing the wheat PCS gene, TaPCS1, showed

an increase in Cd accumulation (Clemens et al., 1999). The finding that an

S. cerevisiae strain that lacked visible vacuoles showed TaPCS1-mediated Cd

tolerance suggests that PCs can act as a cytosolic buffer for Cd and other metal

ions (Clemens et al., 1999). Transcriptional regulation of TaPCS1 was found in

4-day-old wheat seedlings.

In Arabidopsis, Cd stress induces the production of various enzymes involved

in Cys synthesis, an important component of GSH and hence, PCs (Harada et al.,

2002). Glutathione is not only necessary for PC synthesis, but also for Cd·GSH



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transport via YCF1-type ABC transporters. Therefore, overexpression of

g-glutamylcysteine synthetase (g-ECS), a key enzyme in the biosynthetic

pathway of GSH, to increase the biosynthesis of GSH may enhance Cd tolerance.

For example, Indian mustard (Brassica juncea) seedlings overexpressing the

E. coli gene GSH1 (encoding g-ECS) in their chloroplasts had higher levels of

GSH, g-Glu-Cys, and PCs (Zhu et al., 1999b). Transgenics showed increased

tolerance to Cd, and shoot Cd concentrations were 40– 90% higher than that in

the wild type (Zhu et al., 1999b). Xiang et al. (2001) expressed the cDNA

encoding g-ECS (the GSH1 gene, May and Leaver, 1995) under the control of the

CaMV 35S promoter in both the sense and antisense orientations in Arabidopsis.

The resulting plants had GSH levels from 3 to approximately 200% of the level

in wild-type plants. However, Arabidopsis plants with elevated levels of both

g-ECS and GSH did not show increased Cd resistance when exposed to Cd.

Overexpression of glutathione synthetase (GS), another enzyme of the GSH

synthetic pathway, also resulted in higher GSH levels. Zhu et al. (1999a)

overexpressed the GS from the E. coli GSH2 gene in Indian mustard and obtained

transgenic plants accumulating three times more Cd than the wild type when

exposed to Cd. Interestingly, yeast strain Dgsh2 (deficient in GS) had the same

capacity to remove Cd from the medium after 24 h as did control cells, while

strain Dgsh1 (deficient in g-ECS) accumulated about twice as much Cd as

controls after the same time period (Gomes et al., 2002). Perhaps, g-ECS can

bind Cd and be a substrate for YCF1 transporter. Pilon-Smits et al. (2000)

expressed a bacterial GSH reductase in B. juncea targeted to the plastids.

Interestingly, Cd levels in shoots were half as high as those in the shoots of

control plants, while root Cd levels were roughly the same. Transgenic plants had

higher GSH levels than controls both in the presence or absence of Cd. No

difference was observed in plant growth.

(c) Metallothioneins and phytochelatins in tobacco. In early work, Wagner

and Trotter (1982) found that Cd induces a ligand protein in tobacco that binds Cd

in mercaptide bonds. Reese and Wagner (1987) demonstrated the formation of

Cd-induced small peptides in tobacco leaves (cv. KY14) and cells (cv. Wisconsin

38). The peptides displayed properties that differed substantially from those of

animal MTs. Hirt et al. (1990) provided evidence that Cd-binding peptides of

N. tabacum (cv. Xanthi) suspension cells appear to be PCs. Voăgeli-Lange and

Wagner (1990) showed that PC – Cd complexes were sequestered in the vacuole

of mesophyll protoplasts of tobacco plants exposed to Cd. In a later work on

N. rustica, Voăgeli-Lange and Wagner (1996) found that when more than 5 mM

Cd was present in the growth medium, the number of g-ECS repeat units

positively correlated with the Cd concentration in the plant. Wagner (1993)

argued that, at low levels of Cd, as is found in most soils (i.e., up to 0.3 mM), Cd

would mostly be complexed by vacuolar citrate, and it would only be at high

levels of Cd exposure that PCs might play a role. Many argued that GSH might be

sufficient to sequester Cd at low exposure levels. However, this hypothesis is



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V. Options to Reduce the Cadmium Content in Tobacco Leaves

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