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The proteomics of heavy metal hyperaccumulation by plants

Review

Giovanna Visioli,Nelson Marmiroli ?

Department of Life Sciences,University of Parma,Parco Area delle Scienze 11/a,43124,Parma Italy

A R T I C L E I N F O

A B S T R A C T

Article history:

Received 2August 2012Accepted 7December 2012Hyperaccumulators are distinguished from non-hyperaccumulators on the basis of their capacity to extract heavy metal ions from the soil,their more efficient root-to-shoot translocation of these ions and their greater ability to detoxify and sequester heavy metals in the shoot.The understanding of the mechanisms underlying metal ion accumulation has progressed beyond the relevant biochemistry and physiology to encompass the genetic and molecular regulatory systems which differentiate hyperaccumulators from non-hyperaccumulators.This paper reviews the literature surrounding the application of proteomics technology to plant metal hyperaccumulation,in particular involving the elements As,Cd,Cu,Ni,Pb and Zn.The hyperaccumulation process across a number of unrelated plant species appears to be associated with proteins involved in energy metabolism,the oxidative stress response and abiotic and biotic stress.The relevance of transducers of the metal stress response to the phenomenon of hyperaccumulation is summarized.Proteomic data complement the more voluminous genomic and transcriptomic data sets in providing a more nuanced picture of the process,and should therefore help in the identification of the major genetic determinants of the hyperaccumulation phenomenon.

Keywords:

Hyperaccumulator Heavy metal Environment Proteomics Protein biomarkers

Contents

1.What are metallophytes?..................................................134

2.Hyperaccumulators .....................................................134

3.From genomics and transcriptomics to proteomics ....................................134

4.Why proteomics?......................................................134

Proteomic techniques and strategies ............................................1355.1.Sample preparation .................................................1355.2.Quantification and data validation ..............

...........................1355.2.1.Gel and non-gel approaches ........................................1355.2.2.MS-based protein quantification ..........

...........................1376.

State of the art in hyperaccumulator proteomics ......................................1376.1.Plant material and experimental procedures ...................................1376.2.Key steps in hyperaccumulation and the proteins involved

...........................1386.2.1.The root proteome .............................................1386.2.2.The shoot proteome .................

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?Corresponding author at:Parco Area delle Scienze 11/a 43124Parma,Italy.Tel.:+390521905606;fax:+390521906123.E-mail address:marmirol@unipr.it (N.

Marmiroli).

https://www.wendangku.net/doc/0713769116.html,/10.1016/j.jprot.2012.12.006

A v a i l a b l e o n l i n e a t w w w.s c i e n c e d i r e c t.c o m

w w w.e l s e v i e r.c o m /l o c a t e /j p r o t

7.Soil proteomics (140)

7.1.Proteins in the rhizosphere (140)

7.2.Metaproteomics (140)

8.Environmental proteomics (141)

9.Perspectives for proteomics applied to hyperaccumulation:potential protein biomarkers (141)

Acknowledgments (142)

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

1.What are metallophytes?

The term metallophyte refers to plant species able to tolerate phytotoxic levels of heavy metal(typically,As,Cd,Cu,Ni,Pb or Zn)pollution,and so can survive and reproduce in metalliferous soils[1].This definition includes ecotypes of species not normally considered to be metallophytes,which have never-theless adapted to tolerate high levels of heavy metals in the soil,commonly by the expression of phenotypic plasticity[1]. Metallophytes have evolved two exclusive strategies to achieve tolerance,the first involving restricting the entry and/or root-to-shoot translocation of the metal ions,and the second tolerating the presence of heavy metal ions in the body of the plant(so-called“hyperaccumulators”)(Fig.1).In the aerial portion(and particularly the leaf)of hyperaccumulators,the concentration of heavy metal can reach two to three orders of magnitude above the critical level for non-hyperaccumulators, without the appearance of any visible symptom of toxicity [2].The majority of hyperaccumulators are associated with metalliferous soils,but some are also able to survive in non-metalliferous soils[1].

2.Hyperaccumulators

The key trait used to define a metal hyperaccumulator is the threshold concentration of the toxic metal ion which can be tolerated(Table1).Typically,a level of1%of shoot dry mass is considered the threshold for Zn and Mn,0.1%for As,Co,Cr,Cu, Ni,Pb,Sb,Se and Tl,and0.01%for Cd(Table1and[3]).Some450 plant species(equivalent to just0.2%of angiosperm species)have been classified as hyperaccumulators,and around75%of these are tolerant of Ni[1,3–9];only five species to date have been classed as hyperaccumulators of Cd,one of the most toxic of the metal ions found in the soil[3].The taxonomic range of hyperaccumulators is wide,including both tropical and temper-ate species.The genus Alyssum(Brassicaceae),many of whose species are native to southern Europe and western Asia,includes 48species capable of accumulating Ni up to a level of3%of dry shoot mass[5].Since hyperaccumulation has apparently evolved independently several times,presumably driven by environmen-tal pressure,it has been suggested that the trait may carry with it a reduced attractiveness to herbivores and/or pathogens,which are unable to cope with the high level of metal ions in the leaf [10–12].Hyperaccumulators are of practical interest as they potentially provide a means to detoxify polluted soil or to harvest valuable metal from metal-rich soils[13–16].A further current area of interest,at least for those metal ions essential for the human diet,lies in the prospect of transferring the trait to crop plants as a way of combatting dietary mineral deficiencies, particularly for the benefit of populations which are exclusively or primarily vegetarian[17,18].

3.From genomics and transcriptomics

to proteomics

Only a small number of hyperaccumulators has been sub-jected to detailed biochemical,physiological and molecular characterization.The focus in Europe over the past15years has been on Noccaea caerulescens ssp.caerulescens(syn.Thlaspi caerulescens),a hyperaccumulator of Cd,Ni,Pb and Zn[9,19] and on Arabidopsis halleri,a species well adapted to soils heavily polluted by Cd,Pb and Zn[20].Both species are brassicaceous, which is advantageous in the context of their relationship with the standard model dicotyledonous species A.thaliana.The genetic and physiological mechanisms underlying metal hyperaccumulation in N.caerulescens and A.halleri have been thoroughly described in the literature[9,21–29].Surprisingly, it appears that despite a concerted effort to characterize the trait's genetic basis and the plant's transcriptomic response to exposure to metal ions[26,30–40],as yet no specific gene function has been associated with the hyperaccumulator phenotype.This failure may reflect a combination of the rather different level of genomic information associated with the model hyperaccumulator and non-hyperaccumulator species, the inability to produce hybrids between these species and the complexity of the trait itself.Currently genomic sequencing is being extended to the hyperaccumulator N.caerulescens(Prof.M.

G.M.Aarts,Laboratory of Genetics,Wageningen University, personal communication)which may help to identify candi-date gene functions in hyperaccumulation via a comparative approach.

4.Why proteomics?

A comprehensive review of the state of knowledge regarding the proteomics of hyperaccumulation has yet to be published.The supposition is that the plasticity of the hyperaccumulator phenotype reflects an interaction between gene action,protein function and the influence of the environment.It has been established that genetic polymorphism,transcriptional gene regulation and epigenetic control all contribute to the adaptation to various environmental stresses[41].While these factors are undoubtedly also important for specifying the hyperaccumulator phenotype[29],post-transcriptional regulation,protein folding and protein/protein interactions are also likely to contribute to

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the trait's determination.Thus a more global omics approach is needed to generate models to explain the cellular basis of hyperaccumulation [42].The current state of knowledge regard-ing the proteomics of hyperaccumulation is inadequate to understand the role of the large number of proteins involved and the level of cross-talk between different pathways (see Section 6,Table 2).Nevertheless,the increasing sophistication of proteomics developed in model species is making inroads into elucidating the molecular basis of hyperaccumulation.Below,we review current proteomic methodologies appropriate for the identification of key regulators of hyperaccumulation.

Proteomic techniques and strategies

5.1.

Sample preparation

Sample preparation is a crucial step in any proteomic analysis,and plant-based materials are typically harder to process than those from other organisms [43].Most proteins are present at a very low concentration within the cell and many plant cells produce a spectrum of secondary metabolites (such as phenolic compounds,starch,oils and cell wall polysaccharides),some of which compromise the extraction process and/or interfere with subsequent fractionation and other downstream processes [43,44].The rigid plant cell wall is difficult to disrupt,so cell lysis is not as straightforward as it is for animal and bacterial cells.Consequently,the extent of protein loss during tissue homogenization can be significant [45].Pertinent sample preparation depends on the experimental design,the tissue targeted and the subsequent separation methodologies applied.Extraction protocols adopted for hyperaccumulators to date have included TCA-[46–48]or acetone-based [49,50]precipita-tion of homogenate from younger plant tissue,and phenol-based extraction followed by precipitation with ammonium acetate from more mature material plant [51].Extraction solvents have been most commonly based on either MgSO 4[52–54],urea [55]or Tris [56–58].A frequently applied approach has involved an initial pulverization of snap-frozen tissue,

followed by lysis facilitated by sonication,centrifugation to remove debris and a desalting step.

5.2.Quantification and data validation 5.2.1.

Gel and non-gel approaches

In most plant proteomic studies,including those focused on hyperaccumulators,pre-fractionation of the sample prior to mass spectometry (MS)analysis is carried out.This can be achieved via either gel electrophoresis or by certain gel-free techniques.Table 2illustrates the approaches taken by a sample of studies seeking to determine the effect on the proteome of exposure to heavy metals in various hyperaccumulators,pub-lished during the period 2005–2012;some of these have relied on two dimensional electrophoresis (2D-GE)and others on two dimensional liquid chromatography (2D-LC).The majority has adopted the former approach as a preliminary to a MS-based

Table 1–Threshold values applied for the definition of hyperaccumulation in plants.Element

Average range in soils (mg/kg dry weight)Average range in plant tissues (mg/kg dry weight)

Threshold for hyperaccumulators (mg/kg dry weight)As 1–400.009–1.51000=0.1%Cd 1–20.03–0.5100=0.01%Co 8

0.1–101000=0.1%Cr 5–10000.2–11000=0.1%Cu 2–602–20

1000=0.1%Hg <0.1

0.005–0.21000=0.1%Mn 100–40001–70010000=1%Ni 2–2000.4–41000=0.1%Pb 10–1500.1–51000=0.1%Sb <10.3–21000=0.1%Se 1–20.01–0.21000=0.1%Tl 1

0.11000=0.1%Zn

25–200

15–150

10000=1%

Data obtained from citations [3and 29],and references

therein.

Fig.1–Mechanisms underlying metal tolerance in metallophytes.Metal ion distribution/accumulation in a non-hyperaccumulator (left ),and a hyperaccumulator (right ).

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identification(Fig.2,Table2).Given technological improvements with respect to achievable resolution and reproducibility,2D-GE has become the method of choice for separating complex protein mixtures[43].The availability of both broad(typically from pH3to 10)and narrow(1pH unit)range isoelectric focussing gels(which represent the first electrophoretic dimension)has allowed for large numbers of discrete proteins to be distinguished[43]. Nevertheless,some weaknesses remain,specifically relating to the limited capacity to successfully fractionate hydrophobic proteins and glycoproteins,the difficulty in detecting low abundance small peptide molecules,and the risk of quantifica-tion bias when relying on labelling(fluorescent dye or radioac-tivity)rather than optical absorbance.The overwhelming signal generated from the highly abundant proteins1,5bisphosphate carboxylase/oxygenase(RuBisCO)(~40%of the protein content of photosynthetic material),storage seed proteins and a few other housekeeping proteins(e.g.α-tubulin,β-actin;ribosomal pro-teins)can easily conceal those generated from low abundance proteins which migrate to a similar part of the2D-GE gel[59and references within].Some of this problem can be solved relatively simply by first depleting the amount of RuBisCO present in the sample;commercially available products can achieve up to90% depletion from a range of plant materials[60].Although2D-GE has become a fairly robust and straightforward technique, powerful enough to successfully separate as many as10,000 different polypeptides[43],it remains labour-intensive;further-more,gel-to-gel comparisons can be complicated by even minor perturbations in reproducibility[43].The reproducibility of LC-based separation is generally better than that achieved by 2D-GE,an important advantage for comparative proteomics [51,61,62].

Various statistical packages have been developed to facili-tate semi-quantitative comparative proteomics;these include “Progenesis”(Nonlinear Dynamics),“ImageMaster2D Platinum”(Ge Healthcare,Amersham Biosciences),“PDQuest”(Bio-Rad) and“Proteovue”(Eprogen).For2D-GE,the software operates on spot area,while for2D-LC it operates on peak area.Differential labelling with Cy3or Cy5dyes(DIGE technology,Ge Healthcare) greatly improves comparisons between samples.Cy fluores-cence is linear over a wide range of concentration and sensitive down to125pg.The spectrophotometrically based(generally at O.D.214nm)detection of2D-LC peaks is also linear over a wide range,which is advantageous for quantification,and is held to be more reliable than platforms using either dye or radioactive labelling[62].Immunoblotting is commonly performed after separation and identification by MS procedures for data validation[43,45].

5.2.2.MS-based protein quantification

The advent of MS technology has greatly improved the quality and throughput of proteomic data over what can be achieved by electrophoretic or chromatographic methods.It facilitates a more reliable quantification of individual protein species,as well as the recognition of post-translational modifications such as phosphorylation and acetylation,which are impor-tant in cell signalling and certain epigenetic phenomena[63].

5.2.2.1.Relative protein quantification:non-target shot gun proteomics.MS-based shot gun proteomic analysis is a high sample-throughput method designed to detect alterations in protein levels following the imposition of a defined exter-nal treatment.A complex protein sample is digested into oligopeptides,which are identified via LC coupled with MS.The identification of the full proteins is then based on piecing together the oligopeptide sequences in silico[64].Quantification is achieved either through the use of various labelling methods (e.g.,iTRAQ,ICAT,ICPL or SILAC)or by using a variety of label-free methods[63].Most of these techniques have been tried on plant systems[65–68].An example of the integration of data sets derived combining2-D DIGE with iTRAQ to character-ize hyperaccumulator proteomes is given in[51].Both a database-dependent and a database-independent analysis of shot gun derived proteomic data can be undertaken for the identification of differentially abundant proteins[69].The former approach concentrates solely on unambiguously iden-tifiable proteins,while the latter attempts to correlate pheno-type with the presence of peptide precursors sharing the same mass to charge ratio(m/z)as derived from raw MS data. Potential candidates are then taken forward for formal identi-fication either by means of a database search or by de novo interpretation.Recently,this approach was applied for the identification of specific proteins in natural germoplasms of potato[70,71].

5.2.2.2.Absolute protein quantification:selected reaction monitoring(SRM).In SRM,the MS device is pre-programmed to analyse an a priori selected set of targets.SRM(sometimes also referred to as“multiple reaction monitoring”)experiments involve assays conducted using a triple quadrupole instrument designed to detect signals arising from a small set of oligopep-tides,each of which is diagnostic for a specific target protein[72]. As a result,the levels of sensitivity,reproducibility and quanti-tative precision are high.A few examples of SRM analyses targeting plant proteins have been published to date[67,73,74]. Although initially limited to the parallel measurement of only a limited number of targets,the technique's capacity has since been extended to some hundreds of analytes per experiment by innovations in instrumentation and software.As a result,a number of new applications have been proposed[73,75].The volume of data generated by SRM experiments has required the development of appropriate statistical and computational meth-odologies.A limitation in the plant proteomics field remains the relative paucity of prior information,but this short-coming is being addressed through the co-ordination of various inter-national consortia,such as MASC(https://www.wendangku.net/doc/0713769116.html,/ portals/masc/)for A.thaliana,and INPPO(https://www.wendangku.net/doc/0713769116.html,/) more generally.

6.State of the art in

hyperaccumulator proteomics

6.1.Plant material and experimental procedures

A number of proteomic investigations conducted on hyperaccumulators has been based on an analysis of root or shoot samples,although cell cultures have also been assayed [49](Table2).Typically,plants are raised hydroponically,and then exposed to heavy metals for varying periods(from a few hours to60days).Only a single case has featured the analysis of

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plants grown in the field [76].The standard experiment has comprised a comparison between individual plants of a given species,where some have been exposed to heavy metal stress and others not [50,51,54,56,57],although some experiments involving N.caerulescens have compared the performance of accessions of a given species which differ with respect to their hyperaccumulating capacity [53,77,78].It has been recognized that for A.halleri (and by implication more generally),the activity of certain soil micro-organisms can modulate the availability of metal ions,and hence the plant's performance in the soil [55,79],while for Pteris vittata ,a similar role is played by arbuscular mycorrhizae (AM)[46,58].Few studies have considered the combined toxicity of more than one metal [47,55,77,79].A better understanding of the proteomics of hyperaccumulation awaits analyses carried out on purified tissues or organelles,as exemplified in studies surrounding the response to other environmental stresses [80].As a recent application,2D-LC –MS/MS approach has been used in N.caerulescens to investigate the abundance of proteins involved in Zn accumulation in leaf epidermal tissue compared with mesophyll tissue,which is the site of Zn accumulation.This study was performed in combination with Zn speciation in the same tissues with SEC-ICP-MS to identify Zn-binding ligands and mechanisms responsible for Zn hyperaccumulation [110].

6.2.Key steps in hyperaccumulation and the proteins involved

The three key steps underlying the capacity to hyperaccumulate are the control of uptake of metal ions into the root,the loading of ions into the xylem,and their subsequent sequestration and detoxification in the shoot tissue.

6.2.1.The root proteome

Hyperaccumulators are distinguished from non-hyperaccumulators by showing an enhanced rate of metal ion uptake,combined with a low level of sequestration in the vacuoles of the root cells and a high rate of ion translocation (Fig.1)[28,29].The outcome of proteomic analyses of root tissue has been the recognition that the translation of various proteins is differentially regulated by the presence of metal ions (Table 2,Fig.3).

6.2.1.1.Ion uptake and translocation.

The use of a segregat-ing population bred from the cross between two contrasting N.caerulescens accessions has demonstrated that the abundance of specific proteins (particularly in the root)is associated with the accumulation of Zn [78].Similarly,in A.halleri ,the gene hma4,which has been identified as a genetic determinant of hyperaccumulation,is also preferentially expressed in the

root

Fig.2–Common steps used in comparative proteomic analyses of plant hyperaccumulators (here featuring N.caerulescens ).The procedures used for protein extraction are dependent on the tissue targeted and the subsequent separation methodologies applied.(A)“In gel ”separation based on 2D-GE,(B)“gel free ”separation based on 2D-LC.Separation is followed by pattern analysis,tryptic digestion and protein identification via MS analysis.

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[40].The concentration of histidine (as well as that of enzymes involved in its synthesis),which is a major contributor to Ni and Zn loading and chelation,has been shown to be enhanced in the root,but not in the shoot of N.caerulescens,while this behavior was not mirrored in the related non-hyperaccumulator species Thlaspi arvense [81].As yet,there is no example of a proteomic analysis successfully identifying a root transporter protein which responds to heavy metal treatment,or whose abundance differs between a hyperaccumulator and a related non-hyperaccumulator.This failure is consistent with the outcomes of transcriptomic analyses which have repeatedly demonstrat-ed that metal transporter genes are constitutively transcribed in hyperaccumulator species [34,35,82].It has been suggested that there may yet be transporters not currently identifiable using available separation technology,which are up-regulated by the presence of metal ions [43].

6.2.1.2.Stress response and reactive oxygen species (ROS)scavenging.A number of proteins involved in sulphur metab-olism,protection against ROS,the hypersensitive response and xenobiotic detoxification is up-regulated by elevated levels of metal ion in the root of many hyperaccumulators;examples are provided by the Ni hyperaccumulator Alyssum lesbiacum [56]and the Cd hyperaccumulator Brassica juncea [51].Proteins active in ROS scavenging have been shown to be particularly abundant in the root tissue of two accessions of the Cd/Zn hyperaccumulator N.caerulescens ,but less markedly so in a Cd/Zn tolerant,but less efficient accumulator [77].While superoxide dismutase was also up-regulated in the former accessions when the plants were challenged with Zn,the level of ascorbate peroxidase was reduced by the stress [77].Modified forms of this class of proteins were rare in the root tissue,even after a prolonged stress treatment.As the level of stress applied was low enough to allow plant growth to continue (although high enough to induce hyperaccumulation),it was hypothesized that the constitutive expression of the relevant genes permits the effective chelation and sequestration of the metal contami-nants,so that there was no need for the plants to synthesize other proteins [53,56].

6.2.1.3.Cellular metabolism.

The root cell wall has the capac-ity to bind metal ions.A putative glycosyl hydrolase family 18protein has been shown to affect the efficiency of binding in

N.caerulescens [65].This protein is known to participate in cell wall re-assembly and particularly in cell expansion,and its abundance was lower in the root of an accession able to accumulate Cd and Ni than in that of an accession which was a less effective Cd accumulator.Cytosolic glutamine synthe-tase,an enzyme specifically expressed in the root pericycle and involved in ammonium assimilation,is known to be transcriptionally repressed by the presence of excessive concentrations of Cd ion.Its abundance appeared to be correlated with Cd tolerance (but not with Cd accumulation)in N.caerulescens [77].

6.2.2.The shoot proteome

In most hyperaccumulators,the shoot represents the major site of metal sequestration and storage.A number of shoot proteins has shown to be regulated by exposure to heavy metal stress (Table 2,Fig.3)

6.2.2.1.Metal chelators and transporters.

The expression in

the shoot (but not in the root)tissue of metal chelator and transporter proteins can be affected by metal stress.For example,the abundance of both a putative metal transporter ZRT/IRT like protein 8and a metal chelator methallothionein (MT)protein 1B was heightened by Ni stress in a hyperaccumulating accession of N.caerulescens ,but was unaffected in a non-hyperaccumulating accession [53].Similarly,the expression of MT4C (a putative MT)could be correlated with the plant's capacity to accumulate Ni [76].The chelation of trace metals is a vital part of the plant's detoxification machinery.Although a correlation between the level of various MTs and that of metal ions has yet to be established,it is evident that hyperaccumulators do express elevated levels of MT,not just in N.caerulescens ,but also in A.halleri ,Silene paradoxa and Silene vulgaris [83–86].Yeast complementation studies have shown that the A.thaliana genes encoding MT4a and MT4b enhance Zn and Cu tolerance and the accumulation of Zn more effectively than do other MTs [87].The N.caerulescens Ni accumulators tend also to show a significantly higher abundance of certain ABC transporter proteins [76].These proteins are involved in the sequestration of metals into the vacuole,other sub-cellular compartments or into the apoplast [88,89].A comparison between two N.caerulescens ecotypes showing contrasting Zn tolerance and Zn accumulation capacity identified the differential

expression

Fig.3–Classes of protein which change in abundance in hyperaccumulators in response to metal exposure,as identified by proteomic analyses.Shoot proteins indicated by black bars,root proteins by grey bars.The data were obtained from the literature listed in Table 2.

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in the shoot tissue of a homologue of AtMRP10[90],while the level of ATH13was found to be higher in N.caerulescens than in the A.thaliana shoot[39].A possible role in conferring tolerance to Cu,Zn,Cd has been ascribed to ABC transporters expressed in micorrhyzae[91].This broad class of proteins has been promoted as a source of markers for the hyperaccumulation phenotype.

6.2.2.2.Energy and carbohydrate metabolism.A consistent up-regulation of proteins involved in photosynthesis(in particular subunits of the chlorophyll a/b binding protein and a membrane extrinsic subunit of photosystem II(PSBP))has been associated with the hyperaccumulator A.halleri in response to stress imposed by Cd and Zn[55],and P.vittata exposed to As[46]. Similar responses have been noted in Arabis paniculata(a Zn/Cd tolerant hyperaccumulator)exposed to Zn or Cd stress[57],in Helianthus annuus(a hyperaccumulator of Pb[47]),and in the monocotyledonous species Agrostis tenuis(As tolerant and a hyperaccumulator[50]).The up-regulation of these proteins has been assumed to impose an enhanced energy demand,and thus represents a trade-off against the level of global meta-bolism[46,55].The increased energy demand requires the activation of enzymes involved in energy metabolism and the Calvin cycle,for example phosphoribulokinase,fructose-bisphosphate aldolase and the RuBisCO large sub-unit,all of which were up-regulated in hyperaccumulators[46,50,57,79].A Zn and Cd tolerant N.caerulescens accession expressed higher levels of PSBP than did two different less tolerant accessions [77].Several Calvin cycle enzymes(RuBisCO large subunit, glyceraldehyde-3-phosphate dehydrogenase(GAPDH)and sedoheptulose-1,7bisphosphatase)were also shown to be significantly more abundant in the tolerant accession[77].

6.2.2.3.Stress proteins and the antioxidant response.An increase in the presence of proteins associated with xenobiotic/ antioxidant defence and the hypersensitive response has also been identified in the shoot tissue of various hyperaccumulators subjected to heavy metal stress.These proteins include certain aldo/keto reductases,GSTs,thioredoxin,cytochrome P450family members,aspartate aminotransferase,superoxide dismutase and ascorbate peroxidase,which detoxifies peroxides produced during the breakdown of lipids[46,47,53,56,57,77].In a number of bacteria,algae and fungi,GSTs have also been shown to respond to heavy metal(Cd,Zn and Cu)stress[92,93].A proteomic analysis of the Cd hyperacumulating lichen Physca ascendes has suggested that GST has a role in both the detoxification and hyperaccumulation of Cd[94].

6.2.2.4.Defence proteins.A number of plant defensins is up-regulated in hyperaccumulators[49,53,55,76].Defensins are a class of proteins which are involved in the hypersensi-tive reaction and during biotic and abiotic stress in plants[95]. In A.halleri,a pool of defensins is Zn responsive,both at the transcript and at the translational level[96].In N.caerulescens, the level of these proteins appears to be correlated with the capacity to accumulate metal ions[53,76],thus strengthening the hypothesis that the capacity of hyperaccumulators to accumulate metal ions in their aerial portion may have evolved as a defence strategy against herbivores and foliar pathogens[10–12].6.2.2.5.Regulatory and signal transduction proteins.Few data are available relating to the regulatory proteins involved in signal transduction.The identification of such proteins using a proteomic approach is limited by their low abundance.Although numerous efforts have been made to improve detection sensi-tivity,the level of resolution achieved by proteomic analysis remains much lower than that which is achievable at the nucleic acid level,where PCR,hybridization and sequencing have become routine[35–39].Nevertheless,a one-step protein extrac-tion procedure coupled with2D-LC and MS analysis was able,in an analysis of the shoot proteome of a hyperaccumulating N.caerulescens ecotype,to identify a putative Ras related protein (Rab7)and a Myb transcription factor which both responded to Ni stress.Neither of these proteins was expressed in a non-hyperaccumulating ecotype[53].Proteomic data have shown how costly in energy terms the hyperaccumulation of metals can be.The adaptation to hyperaccumulation has therefore been the up-regulation of photosynthesis and the synthesis of proteins to remove the breakdown products.

7.Soil proteomics

7.1.Proteins in the rhizosphere

The rhizosphere is a dynamic micro-environment in which micro-organisms,roots and soil interact with one another[97]. The soil microbial community has emerged as an important component of the hyperaccumulation process[98,99].Interac-tions between the various bacterial communities which can differ with respect to their tolerance of heavy metal ions clearly make a contribution to the capacity of plants to hyperaccumulate,as shown by the involvement of specific microbial proteins in the process[46,55,58,79].The presence of rhizosphere micro-organisms has been positively correlated with the accumulation of both Cd and Zn in the shoot tissue of A.halleri[55,79].These micro-organisms can up-regulate various plant proteins involved in photosynthesis and in the Calvin cycle,and down-regulate both a range of plant defence response proteins and the level of GST.In effect,they relieve the plant of certain defence activity, but only at a metabolic cost to the host[55,79].The presence of AM serves to enhance the availability of minerals and to provide a measure of protection against both biotic and abiotic stress[100–102].The ability to grow in Cd polluted soils is improved by AM through its capacity to increase the availability of phosphorus and to restrict the uptake of Cd[103,104].When P. vittata plants were stressed with As in the presence of AM,it was noted that an aldehyde dehydrogenase(an enzyme involved in ROS scavenging)was up-regulated[46,58]Similarly,exposure to As in the presence of AM resulted in the down-regulation of chaperonin60and glutamine synthetase,involved in protein degradation and of S-adenosylmethionine synthase(assumed to be involved in As methylation)[58].

7.2.Metaproteomics

Studying the protein level interactions occurring between a hyperaccumulator plant and the soil has been termed “metaproteomics”[105].The proteome contributed by the

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soil microbe community may well make a significant contribu-tion to the hyperaccumulation process.The extension of pro-teomics to the soil requires drastic adjustments to be made to the protocols used for protein extraction and sample preparation.Many gaps remain to be filled in this context,some technical and others informational.Thus,for example,the per unit sample quantity of protein in a soil sample is far smaller than that present in a purified microbial culture sample;furthermore,soils contain a range of compounds which can interfere with protein identification (e.g.,humic acids).As yet,very little genomic data has been assembled to allow the easy identification of proteins produced by soil micro-organisms [106].However,it is to be anticipated that the presence (or absence)of specific microbial proteins will eventually be useful as an indicator for positive interactions between the plant root and the soil flora,and hence to be predictive of the hyperaccumulator phenotype.

8.Environmental proteomics

The external environment of the plant has a large effect on the plant proteome,an example are those proteins which act as sensors of environmental perturbation,and which then transmit the signal within the plant to activate the various genetic and metabolic pathways through which the plant's ultimate response is enacted.Protein modifications,in particular phosphorylation,acetylation and glucosylation,are likely to contribute to the phenotypic plasticity necessary for the hyperaccumulation trait [107].In this context,it is important to both compare individuals

of a given species which show contrasting adaptation,and to identify variation in key proteins in plants grown in a natural environment.

While the use of controlled hydroponics grown plants greatly simplifies the experimental set up and allows for the imposition of a precisely defined level of metal stress,this environment is far removed from the real world of hyperaccumulators grow-ing under natural conditions.As an illustration,when a Cd hyperaccumulating ecotype of N.caerulescens was grown in both its native soil and under laboratory conditions,its measured Cd tolerance proved to be greater in the latter case [77].Many plants show a high level of phenotypic plasticity in response to variation in the environment.At the level of the proteome,a study of a Ni hyperaccumulating N.caerulescens ecotype grown in its native environment has shown that differences in accumulation ca-pacity within the population are reflected in the abundance of metal transporters and chelators,proteins related to defence against biotic and abiotic stresses,proteins of general metabolism and regulatory proteins [79].

9.Perspectives for proteomics applied to

hyperaccumulation:potential protein biomarkers

To date,a limited number of attempts has been made to study the proteome of just a handful of hyperaccumulators (N.caerulescens ,B.juncea and A.halleri )subjected to a range of stress agents;nevertheless,these experiments have already led to the recognition of some common proteins,as summarized

Fig.4–Heat map created by TreeView software showing protein levels induced by exposure to metal stress in N.caerulescens ,B.juncea and A.halleri .Data assembled from various sources [51,53,55,63–66],and listed also in Table 2.Fold variation between data was normalized as follows:1–2fold=1,2–3fold=2,3–5fold=3,>5fold=4corresponding to increasing intensity of the red colour.The intensity of the green colour represents equivalent levels of down-regulation.The black squares represent absence of data.

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the heat map shown in Fig.4.The adjustment in the level of expression of some of these proteins has revealed a trade-off between adaptation and energy production.Other common features appear to be an increased synthesis of biotic and abiotic stress response proteins[108]and of ROS scavengers and other detoxifiers of reactive intermediates of the hyperaccumulation response.The nature of the metal stress response transducers used by hyperaccumulators has not been defined as yet, although evidence of their contribute to hyperaccumulation emerge also from proteomic studies[53,65].The hope is that specific proteins will eventually be identified as markers for the hyperaccumulator phenotype,driven by developments in proteomic technology,such as the“targeted analysis”described above in Section5.2.2.Effective markers could find a use in marker assisted selection breeding for enhanced metal uptake in non-hyperaccumulator economically important species [109].

Acknowledgments

We acknowledge the financial support provided by the AGER project(grant2010-0278)and the PRIN2008project“Genotypic and phenotypic variability of a N.caerulescens population adapted to grow on the ophiolitic soil of Monte Prinzera”.The authors thank the University of Parma for the purchase of relevant equipment and access to specialist facilities at its Interdepartmental Measure Centre(CIM).We also appreciate the contribution of COST action FA0905(coordinated by Prof.Bal Ram Singh,Norwegian University of Life Sciences,Norway),and the International Plant Proteomic Organization for the setting up of a network of information and resources for the diffusion of plant proteomics.Thanks also to Dr.Robert Koebner for critically reading the manuscript and supporting for English spelling.

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