Phytoremediation of Contaminated Soil and Water - Chapter 12 doc

12 METAL TOLERANCE IN PLANTS: THE ROLE OF PHYTOCHELATINS AND METALLOTHIONEINS Peter Goldsbrough CONTENTS Introduction Phytochelatins Genetic Analysis of Cadmium Tolerance Metallothionein

12 METAL TOLERANCE IN

PLANTS: THE ROLE OF PHYTOCHELATINS AND METALLOTHIONEINS

Peter Goldsbrough

CONTENTS

Introduction

Phytochelatins

Genetic Analysis of Cadmium Tolerance

Metallothioneins

The Arabidopsis MT Gene Family

RNA Expression of Arabidopsis MT Genes

Expression of Plant MT Proteins

Metal Binding Properties of MTs

Are MTs Required for Metal Tolerance in Plants?

Manipulation of Metal Ligands for Phytoremediation

References

INTRODUCTION

The successful development of phytoremediation as a method for treatment of contaminated sites depends in part on identifying plant material that is well adapted

to the environmental conditions that prevail at such sites There will be a great deal

of variation in these sites, from those that are severely degraded and need significant modification and amendment before any plants will grow, to others that have rela-tively good conditions apart from the presence of toxic contaminants Developing both cultivation practices and plant varieties for these environments are major chal-lenges that must be addressed if phytoremediation is to develop into a widely adopted technology for the restoration of polluted environments As with virtually all agro-nomic and horticultural practices, plant varieties will have to be selected that are well adapted and able to perform under specific conditions Selection of suitable varieties may also be complemented by biotechnological approaches, including gene transfer, to further improve the capacity of plants to function under these conditions

Remediation of metal-contaminated sites by plants depends on metal uptake by roots and transport of toxic metals to shoots for subsequent harvest and removal One trait that is of great significance to these physiological processes is the ability

of plants to tolerate the metals that are being extracted from the soil This is complicated by the fact that several metals are essential for normal plant growth but are toxic at excessive concentrations, and that many nonessential metals have chem-ical properties similar to those of essential metals Plants are not unique in having

to protect themselves against the toxic effects of metals Thus, a variety of tolerance and resistance mechanisms have evolved, including exclusion or active efflux sys-tems to minimize the cellular accumulation of metals While these are effective protective strategies, they result in low concentrations of metal ions in the organism, precisely the opposite outcome of that desired for phytoremediation, where the goal

is to maximize metal accumulation in plant material Therefore, physiological mech-anisms that are based on tolerance rather than avoidance of metals are likely to be important for phytoremediation, as these will allow plants to survive (and hopefully thrive) while accumulating high concentrations of metals This chapter will address the role of two types of metal ligands, phytochelatins and metallothioneins, in tolerance of plants to heavy metals

PHYTOCHELATINS

Phytochelatins (PCs) were first identified as Cd-binding peptides in

Schizosaccha-romyces pombe and subsequently shown to perform a similar function in a number

of plant species (for recent reviews, see Rauser, 1995; Fordham-Skelton et al., 1997b) Phytochelatins are comprised of a family of peptides with the general structure (γ-GluCys)n-Gly, where n = 2 to 11 Similar (γ-GluCys)n peptides with carboxy-terminal amino acids other than Gly have been identified in a number of plant species, but it is likely that these serve the same function as PCs PCs appear

to be ubiquitous in the plant kingdom, having been shown to accumulate in a wide

variety of species (Gekeler et al., 1989) PCs are also found in fungi other than S.

pombe, including Candida glabrata, an opportunistic pathogen of humans that

infects immunocompromised patients (Mehra et al., 1988)

The γ-carboxamide linkage between glutamate and cysteine indicates that PCs are not synthesized by translation of a mRNA, but are instead the product of an enzymatic reaction A number of studies demonstrated that glutathione ( γ-GluCys-Gly, GSH) is the substrate for synthesis of PCs (the pathway of PC synthesis is illustrated in Figure 12.1) An enzyme activity that catalyzes the formation of PCs from GSH has been described in cell-free extracts from a number of plant species (Grill et al., 1989; Klapheck et al., 1995; Chen et al., 1997) This enzyme, PC synthase, transfers γ-GluCys from GSH to an acceptor GSH to produce (γ-GluCys)2 -Gly (PC2); the same enzyme can add additional γ-GluCys moieties, derived from either GSH or PCs, to PC2 to produce larger PC peptides PC synthase activity is dependent on the presence of one of a number of free metal ions, e.g., Cd2+, Zn2+,

Ag+ Chelation of metal ions, for example, by newly synthesized PCs, inactivates

PC synthase, thereby providing a simple method to regulate the synthesis of PCs The enzyme is constitutively expressed in plant roots and stems (Chen et al., 1997)

and in plant cells growing in culture (Grill et al., 1989), perhaps providing a constant protective mechanism against heavy metal toxicity In spite of the fact that PC synthase was reported to have been purified from plant cells several years ago (Grill

et al., 1989), the gene encoding this protein has proved elusive It is likely that this gene will finally be cloned either by complementation (in fungi) or positional cloning

(in Arabidopsis), using Cd-sensitive mutants that lack PC synthase activity.

Phytochelatin synthesis in plant cells and PC synthase activity can be induced

by a wide variety of metal ions, and PCs are able to bind a number of metal ions

in vitro through thiolate bonds However, the only metal-PC complexes that have

been isolated from plants contain ions of Cd, Cu, or Ag (Maitani et al., 1996)

Cd-PC complexes have been extensively studied and are classified as either high or low molecular weight (HMW or LMW) complexes Two important differences distin-guish these complexes: cellular location and incorporation of cadmium sulfide The HMW complexes accumulate in the vacuole and contain CdS, perhaps in the form

of a microcrystalline structure (Dameron et al., 1989) Partition of HMW complexes

in the vacuole provides an effective method to separate Cd from the majority of metabolic processes Incorporation of CdS in HMW complexes increases the amount

FIGURE 12.1 Synthesis of phytochelatins and formation of HMW Cd-PC complexes The

left column shows defined steps in the PC detoxification system used by plants and some fungi The middle column shows the proteins and genes that are responsible for these activities

CAD1 and CAD2 are genes identified in Arabidopsis, whereas HMT1, ADE2, ADE6, ADE7,

and ADE8 have been identified in Schizosaccharomyces pombe Comments on these steps are

given in the right column

of Cd that is sequestered per molecule of PC HMW Cd-PC complexes are also

more stable, requiring a lower pH to dissociate than LMW complexes S pombe

mutants that do not make HMW Cd-PC complexes are sensitive to Cd (Mutoh and Hayashi, 1988), demonstrating the importance of vacuolar compartmentation for Cd tolerance Vacuolar HMW Cd-PC complexes appear to be the final step in cellular detoxification of Cd, with no evidence for further metabolism of PCs or export of Cd

GENETIC ANALYSIS OF CADMIUM TOLERANCE

A large number of physiological studies indicate that PCs are critical for Cd tolerance

in plants Indirect inhibition of PC synthesis with buthionine sulfoximine, an inhib-itor of GSH synthesis, reduces Cd tolerance, whereas an exogenous supply of GSH increases both PC synthesis and Cd tolerance Cell lines selected for increased Cd tolerance do not exclude Cd from cells but instead accumulate Cd with essentially all of the Cd present in the form of Cd-PC complexes However, the identification and characterization of Cd-sensitive mutants has clarified the role of PCs in tolerance

of plants not only to Cd but also to other metals Genetic analysis has also contributed

to our understanding of cellular partitioning of Cd-PC complexes

Cadmium-sensitive mutants of Arabidopsis thaliana were identified by Howden

and Cobbett (1992), initially using a screen for root growth inhibition on Cd-containing medium Two loci were identified that are essential for normal Cd

toler-ance, CAD1 and CAD2 Mutants at either locus had a number of similar character-istics, including reduced uptake of Cd and lower accumulation of PCs cad1 mutants

had normal levels of GSH but were deficient in PC synthase activity (Howden et

al., 1995b), whereas the single cad2 mutant had a reduced level of GSH but normal

PC synthase activity (Howden et al., 1995a) These mutants have confirmed the importance of PCs for Cd tolerance in plants It was initially proposed that PCs provided tolerance to all heavy metals in plants based on the ability of many metals

to induce PC synthesis (Grill et al., 1987) However, PC-deficient cad mutants are

hypersensitive to cadmium, mercury, and lead (Howden and Cobbett, 1992; Chen and Cunningham, personal communication), but have essentially normal levels of tolerance to other metals, including copper and zinc This demonstrates that PCs are not required for tolerance to all heavy metals and may be restricted to detoxification

of nonessential metals A similar spectrum of metal tolerance has been described

for GSH-deficient (and, therefore, PC-deficient) mutants of S pombe (Glaeser et

al., 1991)

Genetic complementation of Cd-sensitive mutants of S pombe has identified

genes that are involved in the accumulation of HMW vacuolar Cd-PC complexes (Figure 12.1) HMT1 encodes a vacuolar membrane ABC-type transporter that can

transport both PCs and LMW Cd-PC complexes from the cytoplasm into the vacuole (Ortiz et al., 1992, 1995) Mutants that lack this transporter do not accumulate HMW complexes and are Cd-sensitive A similar vacuolar PC transport activity has been identified in plants (Salt and Rauser, 1995), indicating that not only PC synthesis but also cellular compartmentation of the PC detoxification system is conserved between plants and fungi The second group of genes identified by complementation

of S pombe mutants was in the adenine synthesis pathway (Speiser et al., 1992).

This pathway is believed to generate the sulfide that is incorporated into the HMW complexes, starting from cysteine sulfinate (Juang et al., 1993) While plants clearly accumulate similar HMW complexes (Reese et al., 1992), the pathway for sulfide

production is unknown and may be derived in the same manner as in S pombe.

Analysis of Cd-sensitive mutants has made a central contribution to our under-standing of Cd tolerance in plants and fungi There are likely to be several other genes that could be identified using the same approach, and this should be an objective for future research

METALLOTHIONEINS

The discovery of PCs in plants led to the proposal that plants do not possess metallothionein (MT) proteins, i.e., gene-encoded, cysteine-rich proteins translated from mRNAs, but instead utilized PCs to fulfill the functions of metal homeostasis and detoxification (Grill et al., 1987) However, only 2 years after the first reports

of PCs in plants, Lane et al (1987) purified the Ec protein from wheat embryos and demonstrated that the amino acid sequence of this protein was consistent with that

of an MT and that this protein bound Zn2+ This was followed by the cloning of genes that encoded MT-like proteins from several plant species While the functions

of these MT genes are still unknown, it is clear that plants are equipped with at least two ligands that use cysteine coordination of metals, namely PCs and MTs Because

this is the most extensively documented gene family, the Arabidopsis MT gene

family will be used as a model to discuss the structure, expression, and possible function of MTs in plants Studies on MTs from other species will be discussed where they add to the overall view of the function of plant MTs

THE ARABIDOPSIS MT GENE FAMILY

The first Arabidopsis MT gene that was cloned in this laboratory (MT1a) was

identified while screening a library for cDNAs representing transcripts that were induced by ethylene (Zhou and Goldsbrough, 1994) A number of other plant MT genes had already been cloned using differential screening procedures to identify genes expressed in particular tissues or under specific environmental conditions The frequent identification of MT genes in this type of screen indicates that at least some plant MT genes are expressed at relatively high levels in terms of RNA abundance

Using the cDNA for MT1a, and the sequence of another Arabidopsis MT gene in

the Genbank database (now called MT2a), homologous genomic DNA sequences were cloned and characterized This revealed the presence of at least five MT genes

in the Arabidopsis genome (Zhou and Goldsbrough, 1995) More recently, the

Arabidopsis EST database and genome sequencing project have revealed the

pres-ence of three additional MT genes The predicted amino acid sequpres-ences of these genes are shown in Figure 12.2 Arabidopsis MT genes are placed in four categories

based on sequence similarity and relationship with MT genes from other plant species With the exception of MT3, each of the other classes contains two active genes Additionally, there is at least one pseudogene, MT1b (Zhou and Goldsbrough, 1995)

The Arabidopsis genome is normally regarded as a model of simplicity, but there

are other examples of large gene families in this species, including those for β-tubulins and chlorophyll a/b binding proteins Metallothioneins in animals are typ-ically encoded by a gene family of varying complexity Is the extensive MT gene

family in Arabidopsis representative of other plant species? Examples of each of the classes present in Arabidopsis have been found in at least one other species The

Arabidopsis MT1 class is homologous to Type 1 plant MTs in the classification

proposed by Robinson et al (1993) Twelve cysteine residues in Type 1 MTs are present as Cys-X-Cys motifs in two distinct domains at the amino- and

carboxy-termini of these proteins Arabidopsis and Brassica napus MT1 proteins are

distin-guished from other Type 1 MTs by having a “spacer” of only 10 amino acids separating the two cysteine domains (Buchanan-Wollaston, 1994), compared to approximately 45 amino acids in other Type 1 MTs However, it is likely that these

MT genes have a common progenitor given the conservation of both the cysteine

residues and the position of the single intron in Type 1 MT genes Arabidopsis MT2

genes are similar to Type 2 plant MTs, where the first pair of cysteines are arranged

as CysCys

Arabidopsis MT3 was found in a search of the Arabidopsis EST database

(Murphy et al., 1997) This gene is present as a single copy in the Arabidopsis

genome (Bundithya and Goldsbrough, unpublished observations) Homologous genes have been described from kiwi fruit and rice The final class of MT genes in

Arabidopsis, MT4, is related to the wheat Ec genes that are expressed during embryo development cDNAs for two genes with homology to wheat Ec MTs were sequenced from a library prepared from RNA from dry seeds

There is now evidence that other species contain more than one class of MT gene For example, maize has genes encoding a Type 1 MT, expressed primarily in roots (de Framond, 1991), and a homolog of the Ec MT that is expressed in seeds (White and Rivin, 1995) Gene families encoding a single class of MT protein have been characterized in tomato (Whitelaw et al., 1997) and cotton (Hudspeth et al.,

1996) Therefore, it is likely that the size of the Arabidopsis MT gene family is not

FIGURE 12.2 Amino acid sequences predicted for Arabidopsis MTs These are predicted

from the DNA sequences of Arabidopsis MT genes that are known to be expressed Cysteine

residues are in bold The four classes of MT genes are based on similarity to each other and

to MT genes identified in other plant species Note that the protein sequences for members

of the MT4 class do not initiate with a methionine because the cDNAs encoding these proteins are not full length

unusual but merely a consequence of the effort put into understanding the structure and content of this species’ genome

RNA EXPRESSION OF ARABIDOPSIS MT GENES

The frequency with which MT genes have been isolated from plant cDNA libraries

in various differential screening experiments indicates that many MT mRNAs are

expressed at relatively high levels Most of the Arabidopsis MT genes that are

expressed in vegetative tissues have been sequenced several times in assembling the

Arabidopsis EST database For example, more than 40 cDNAs corresponding to

MT3 have been identified The overall pattern of RNA expression of Arabidopsis

MT genes is shown in Table 12.1 MT1 RNA is more abundant in roots than in leaves, whereas RNAs for MT2 and MT3 are expressed at higher levels in leaves than roots (Zhou and Goldsbrough, 1994, 1995; Bundithya and Goldsbrough,

unpub-lished observations) RNA hybridization experiments indicate that the Arabidopsis

MT4 genes are only expressed during seed development (Dandelet, Bundithya, and Goldsbrough, unpublished observations) This is supported by the lack of any ESTs corresponding to MT4 in cDNA libraries prepared from vegetative tissues

Expression of some Arabidopsis MT genes can be induced by metals, notably

copper In the MT2 family, the level of MT2a RNA increases when seedlings are exposed to copper ions (Zhou and Goldsbrough, 1995; Murphy and Taiz, 1995) Copper induction of MT RNA expression has been demonstrated for other MT genes,

both in Arabidopsis and other species, suggesting a role for MTs in an adaptive

response to copper (Hsieh et al., 1995; Robinson et al., 1993) However, two obser-vations suggest caution is warranted with this interpretation First, many other MT genes have been shown not to be induced by copper or other metals This may be

TABLE 12.1

Summary of RNA Expression of Arabidopsis MT Genes

RNA Expression Gene Seedling Roots Leaves Flowers Seeds

Copper Induction (Tissue)

MT1a/c +++ +++ + ++ (leaves) MT2a + + ++ + +++ (seedlings) MT2b ++ + ++ + + (seedlings) MT3 ++ + ++ nd nd + (leaves) MT4 ++ nd

Note: The relative level of expression of RNAs from each MT gene is indicated The tissues

in which copper induction of MT RNAs have been observed are also indicated Gene-specific probes have not been used to examine specifically the expression of MT1a and MT1c.

nd = not determined

the result of examining the expression of MT genes that are not metal regulated in these species, or using conditions where the MT genes are already expressed at a high level and are refractory to further induction Second, MT RNA expression can

be induced by a variety of other environmental and developmental conditions, including heat shock, aluminum stress, nutrient starvation, senescence, and

abscis-sion (reviewed by Fordham-Skelton et al., 1997b) Therefore, while Arabidopsis MT

genes have been shown to be regulated by copper, it is not yet clear if copper induction can be separated from a general stress response Answers to these questions will come from a detailed analysis of the transcriptional regulation and promoter activities of a number of MT genes One approach is to study reporter gene expres-sion driven by MT gene promoters Fordham-Skelton et al (1997a) have shown that the pea PsMTA promoter is active in many tissues in transgenic Arabidopsis,

includ-ing leaves, cotyledons, and floral organs, but is maximally expressed in roots, in agreement with RNA hybridization results A promoter from a cotton MT gene is also highly expressed in roots, notably the root apex (Hudspeth et al., 1996) Com-prehensive analysis of the tissues where individual MT genes are expressed and of the conditions that modulate this expression should provide some insight into the functions of MT genes in plants

EXPRESSION OF PLANT MT PROTEINS

The first evidence that plants synthesized MT proteins, in addition to PCs, came from the work of Lane et al (1987), who demonstrated not only that the wheat Ec protein bound Zn2+, but that its amino acid sequence was consistent with that of an

MT Ec proteins can bind approximately 5% of the zinc in a seed, but they are not expressed in vegetative tissues In spite of the large number of genes encoding MTs that have been cloned, there has, until recently, been no information on the expression

of these “nonseed” MT genes at the protein level Results of Murphy et al (1997) may help explain some of the difficulties encountered in trying to identify MT proteins in plants Low molecular weight, copper-binding proteins were purified

from various Arabidopsis tissues Amino acid sequences of tryptic fragments

obtained from some of these proteins corresponded perfectly with those predicted from the sequences of MT1a/c, MT2a, MT2b, and MT3, providing a categorical demonstration that these MT genes are indeed expressed as proteins If the protein extracts were exposed to oxygen during the first steps of the isolation procedure,

MT proteins could not be recovered The sensitivity of these proteins to oxygen likely accounts for the difficulty in isolating MTs from plants

In addition to demonstrating the presence of MTs in vegetative tissues, Murphy

et al (1997) used antibodies raised against MT-GST fusion proteins to show that expression of MT1 and MT2 proteins reflected the RNA expression of these genes

in terms of tissue specificity and copper induction This correspondence between RNA and protein expression does not rule out the possibility of more complex regulation of the expression of these genes through a number of post-transcriptional mechanisms

METAL BINDING PROPERTIES OF MTS

The wheat Ec protein was identified as a Zn-binding protein, and Arabidopsis MTs

were purified using copper-affinity chromatography However, because of the diffi-culties in purifying MTs from plants, there is a lack of information about the metals

that are bound to MTs in vivo An alternative approach to address this question has

been to express plant MT genes in a number of microbial hosts and either directly assess the metal binding properties of these proteins or examine the ability of plant MTs to confer metal tolerance

When expressed in E coli, either as the native protein or as a fusion protein,

the pea MT was shown to bind Cu, Cd, and Zn ions In its native form, i.e., not a fusion protein, the MT was cleaved within the spacer region, giving rise to two cysteine-rich peptides which could function as independent metal ligands (Kille et al., 1991) Similar processing of MTs in plants might contribute to the difficulties encountered in trying to purify these proteins from plants The affinity of various metals for the MT fusion proteins was assessed by examining metal dissociation at low pH The pea MT had the highest affinity for Cu and the lowest for Zn (Tommey

et al., 1991)

A number of microbes contain MTs that are required for metal tolerance Mutant strains that lack MTs and, therefore, have reduced tolerance to metals have been used as transformation hosts to examine the functional properties of plant MTs

Arabidopsis MT1a and MT2a proteins were expressed in a yeast strain in which one

of its endogenous MT genes, CUP1, had been deleted Constitutive expression of the Arabidopsis MTs restored copper tolerance and increased cadmium tolerance

(Zhou and Goldsbrough, 1994) Similarly, the MT2a protein was able to restore

some degree of zinc tolerance to a Synechococcus mutant that lacked its own zinc

MT (Robinson et al 1996) These experiments have established that Arabidopsis MTs indeed function in vivo as metal of binding proteins.

Differences in metal tolerance between yeast transformants expressing

Arabi-dopsis MT1 or MT2 may reflect differences in the affinity of these MTs for metals,

raising the possibility that the complexity of the Arabidopsis MT gene family is

necessary to deal with a variety of metals The arrangement of cysteine residues in different plant MTs may affect the metal-binding specificity of these proteins Important objectives for future research are to identify the metal ions that bind to

MTs in vivo and to determine the intracellular localization of these proteins Answers

to these questions should contribute to an understanding of the functions of MTs in plants

ARE MTS REQUIRED FOR METAL TOLERANCE IN PLANTS?

Phytochelatin-deficient mutants of Arabidopsis have essentially normal tolerance to

Cu and Zn, indicating that PCs are not required for tolerance to these metals and that there must be other mechanisms to provide tolerance to these metals in plants MTs are one candidate to fill this role, and a number of observations support this

hypothesis As discussed above, plant MTs can bind Cu and Zn and function in vivo

to provide tolerance to these metals in other organisms Expression of MT genes can be induced by Cu, and expression of MT2 RNA is elevated in a Cu-sensitive

mutant of Arabidopsis, cup1, which accumulates higher concentrations of Cu (van Vliet et al., 1995) In a survey of Arabidopsis ecotypes for differences in metal

tolerance, Murphy and Taiz (1995) demonstrated a positive correlation between Cu tolerance of seedlings, measured as root growth after transfer to a Cu-supplemented medium, and expression of MT2 RNA This suggests that expression of at least some MTs is important for Cu tolerance More direct evidence will have to await studies on transgenic plants with altered expression of MT genes and detailed analysis of mutants with altered tolerance to Cu, Zn, and other metals

MANIPULATION OF METAL LIGANDS FOR

PHYTOREMEDIATION

Plants that are selected and developed for phytoremediation will need to have a number of advantageous physiological traits, including tolerance of metals and other environmental conditions at the contaminated site, enhanced uptake and transport

of metals, and sequestration of metals in shoot tissues Manipulating the expression

of PCs and MTs might play a part in one or more of these traits However, there is only limited information available about the best targets for this approach or the likely outcome of such efforts

While cloning a gene for PC synthase has not yet been accomplished, altering the expression of this gene in plants may not have a significant impact on metal tolerance The enzyme is constitutively expressed in many tissues, and its activity

is regulated by free metal ions However, genes for enzymes of GSH synthesis may hold more promise Increased activity of γ-GluCys synthetase in selected Cd-tolerant tomato cells could increase GSH and PC synthesis and contribute to Cd tolerance (Chen and Goldsbrough, 1994) Genes encoding γ-GluCys synthetase and GSH synthetase have been isolated from tomato Surprisingly, neither of these genes shows any change in RNA expression in plants or cells that are exposed to Cd (Kovari and Goldsbrough, unpublished observations) Regulation of these genes may occur at a post-translational level, but this remains to be demonstrated The potential applica-tion of gene transfer to manipulating metal tolerance has been indicated by showing that expression of tomato γ-GluCys synthetase could restore some degree of Cd

tolerance to the cad2 Arabidopsis mutant (Kovari, Cobbett, and Goldsbrough,

unpub-lished observations) However, this gene did not increase Cd tolerance of wildtype plants, perhaps due to an inadequate level of expression or other regulatory problems The vacuolar sequestration pathway may provide another target to increase metal

tolerance Increasing the expression of the HMT1 transporter in S pombe resulted

in Cd hypertolerance (Ortiz et al., 1992) A plant homolog of this transporter has not yet been cloned There are a number of possible approaches to altering PC metabolism that might contribute to increased metal tolerance, and in general there

is a positive correlation between PC synthesis and Cd accumulation However, the outcome of such experiments is uncertain because of our lack of understanding of how GSH synthesis, PC synthesis, and vacuolar compartmentation are regulated