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“fpls-03-00310” — 2013/1/24 — 17:14 — page 1 — #1

REVIEW ARTICLEpublished: 25 January 2013

doi: 10.3389/fpls.2012.00310

Contribution of proteomic studies towards understandingplant heavy metal stress responseZahed Hossain1* and Setsuko Komatsu 2*

1 Department of Botany, West Bengal State University, Kolkata, West Bengal, India2 National Institute of Crop Science, Tsukuba, Japan

Edited by:

Pingfang Yang, Wuhan BotanicalGarden, Chinese Academy ofSciences, China

Reviewed by:

Hans-Peter Mock, Institute of PlantGenetics and Crop Plant Research,GermanyZhulong Chan, Wuhan BotanicGarden, Chinese Academy ofSciences, China

*Correspondence:

Setsuko Komatsu, National Instituteof Crop Science, Kannondai 2-1-18,Tsukuba 305-8518, Japan.e-mail: [email protected];Zahed Hossain, Department ofBotany, West Bengal State University,Kolkata 700126, West Bengal, India.e-mail: [email protected]

Modulation of plant proteome composition is an inevitable process to cope with theenvironmental challenges including heavy metal (HM) stress. Soil and water contaminatedwith hazardous metals not only cause permanent and irreversible health problems, but alsoresult substantial reduction in crop yields. In course of time, plants have evolved complexmechanisms to regulate the uptake, mobilization, and intracellular concentration of metalions to alleviate the stress damages. Since, the functional translated portion of the genomeplays an essential role in plant stress response, proteomic studies provide us a finer pictureof protein networks and metabolic pathways primarily involved in cellular detoxification andtolerance mechanism. In the present review, an attempt is made to present the state ofthe art of recent development in proteomic techniques and significant contributions madeso far for better understanding the complex mechanism of plant metal stress acclimation.Role of metal stress-related proteins involved in antioxidant defense system and primarymetabolism is critically reviewed to get a bird’s-eye view on the different strategies of plantsto detoxify HMs. In addition to the advantages and disadvantages of different proteomicmethodologies, future applications of proteome study of subcellular organelles are alsodiscussed to get the new insights into the plant cell response to HMs.

Keywords: antioxidant, heavy metal, HSPs, phytochelatins, proteomics, PR protein

INTRODUCTIONHigh-throughput OMICS techniques are extensively beingexploited in recent times to dissect plants molecular strategies ofheavy metals (HMs) stress tolerance. Plants growing in HMs con-taminated environment have developed coordinated homeostaticmechanisms to regulate the uptake, mobilization, and intracellularconcentration of toxic metal ions to alleviate stress damages. Asthe functional translated portion of the genome play a key role inplant stress response, proteomic studies provide us a finer pictureof protein networks and metabolic pathways primarily involvedin cellular detoxification and tolerance mechanism against HMtoxicity.

By definition, elements having specific gravity above five areconsidered as HMs. Nevertheless, the term HM commonly refersto toxic metals, e.g., cadmium (Cd), copper (Cu), chromium (Cr),lead (Pb), zinc (Zn) as well as hazardous metalloids viz., arsenic(As), boron (B), which exert negative effects on plant growth anddevelopment (Hossain et al., 2012a).

Most of the HMs get their entry into plant root system viaspecific/generic ion carriers or channels (Bubb and Lester, 1991).The lack of specificity of transporters that are primarily involvedin uptake of essential elements such as Zn2+, Fe2+, and Ca2+

Abbreviations: CBB, coomassie brilliant blue; 2-DE, two-dimensional polyacry-lamide gel electrophoresis; GS, glutamine synthetase; GSH, glutathione; GST,glutathione S-transferase; IEF, isoelectric focusing; LC, liquid chromatography;MS, mass spectrometry; MTs, metallothioneins; PCs, phytochelatins; pI, isoelec-tric point; PR, pathogenesis related; ROS, reactive oxygen species; SOD, superoxidedismutase.

allow the entry of Cd2+, Pb2+ (Welch and Norvell, 1999; Perfus-Barbeoch et al., 2002). Once HM ions enter the cell, cellularfunctions are affected by a wide range of actions. The negativeimpact of HM includes binding of HM ions to sulfhydryl groupsof proteins, replacement of essential cations from specific bindingsites, leading to enzyme inactivation and production of reactiveoxygen species (ROS), resulting in oxidative damages to lipids,proteins and nucleic acids (Sharma and Dietz, 2009).

Over the last decade, extensive research on plants response toHM stress has been conducted to unravel the tolerance mecha-nism. Genomics technologies have been useful in addressing plantabiotic stress responses including HM toxicity (Bohnert et al.,2006). However, changes in gene expression at transcript levelhave not always been reflected at protein level (Gygi et al., 1999).An in-depth proteomic analysis is thus of great importance toidentify target proteins that actively take part in HM detoxificationmechanism.

Plant response to HM stress has been reviewed extensively overthe past decade (Sanita Di Toppi and Gabbrielli, 1999; Cobbett,2000; Ma et al., 2001; Cobbett and Goldsbrough, 2002; Hall, 2002;Maksymiec, 2007; Sharma and Dietz, 2009; Verbruggen et al., 2009;Yang and Chu, 2011; Hossain et al., 2012a). However, review arti-cles on application of proteomics in analyzing cellular mechanismfor HM tolerance are limited (Ahsan et al., 2009; Luque-Garciaet al., 2011; Villiers et al., 2011).

Current review represents the state of art of recent devel-opments in proteomic techniques and significant contributionsmade so far to strengthen our knowledge about plants HM-stress

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Hossain and Komatsu Plant heavy metal stress proteomics

response cascade at protein level. Special emphasis is given tohighlight the role of metal stress-related proteins engage in HMions sequestration, antioxidant defense system, and primarymetabolism for deeper understanding of coordinated pathwaysinvolve in detoxification of HM ions within plant cells. Fur-thermore, future applications of proteome study of subcellularorganelles are discussed to get the new insights into the plant cellresponse to HMs.

QUANTITATIVE PROTEOMIC TECHNIQUES USED FORANALYSIS OF HM-RESPONSIVE PROTEINSConventional two-dimensional gel electrophoresis (2-DE)approach coupled with protein identification by mass spectrome-try (MS) has been the most widely used proteomic technique forinvestigation of HM-induced alteration of plant proteome com-position (Table 1). Protein extraction and purification from theHM-stressed tissue is the most crucial step in 2-DE approach,as the amount and quality of the extracted proteins ultimatelydetermine the protein spot number, resolution, and intensity. Phe-nolic compounds, proteolytic and oxidative enzymes, terpenes,pigments, organic acids, inhibitory ions, and carbohydrates aresome common interfering substances present in recalcitrant planttissues. Inferior 2-D separation results due to proteolytic break-down, streaking, and charge heterogeneity. Proteomic studies onplant response against HM stress have revealed that trichloroaceticacid/acetone precipitation (Patterson et al., 2007; Zhen et al., 2007;Kieffer et al., 2008; Alves et al., 2011; Hossain et al., 2012b,c) andphenol-based (Bona et al., 2007; Alvarez et al., 2009; Vannini et al.,2009; Lee et al., 2010; Ritter et al., 2010; Rodríguez-Celma et al.,2010; Ahsan et al., 2012; Sharmin et al., 2012) protocols are theeffective protein extraction methods for obtaining high qual-ity proteome map. Nevertheless, phenol-based method is themost appropriate in extracting glycoproteins, and produce high-resolution proteome map for recalcitrant plant tissues (Saravananand Rose, 2004; Komatsu and Ahsan, 2009).

As compared to classical staining procedure of 2-DE gel usingCBB or silver staining, advanced fluorescence two-dimensionaldifference gel electrophoresis (2-D DIGE) proteomic approachis now being used which allows comparison of the differentiallyexpressed proteins of control and HM-stressed tissue on one singlegel (Kieffer et al., 2008; Alvarez et al., 2009). DIGE is basically a gel-based method where proteins were labeled with fluorescent dyes(CyDyes – Cy2, Cy3, and Cy5) prior to electrophoresis. With theadvancement of technology multiplexed isobaric tagging (iTRAQ)of peptides has allowed comparative, quantitative analysis of mul-tiple samples. This second generation gel free proteomic approachhas been well exploited for gaining comprehensive understandingof plants response to Cd and B (Patterson et al., 2007; Alvarez et al.,2009; Schneider et al., 2009).

PLANT STRATEGIES OF HM TOLERANCEIn course of time, higher plants have evolved sophisticated mech-anisms to regulate the uptake, mobilization, and intracellularconcentration of HM ions (Figure 1). Apart from the plasmamembrane exclusion method, the most common way to protectthe cell from the adverse effects of HMs includes synthesis of mem-brane transporters and thiol-containing chelating compounds

FIGURE 1 | Schematic illustration of various cellular mechanisms for

mitigating heavy metal (HM) stress. Information about highlightedproteins gathered from published proteomic articles related to plantHM-toxicity. Up and down arrows indicate HM-induced increase anddecrease protein abundance respectively. ATPase β, ATP synthase subunitbeta; AH, aconitate hydratase; AsA-Glu, ascorbate glutathione; APX,ascorbate peroxidase; ACC, 1-aminocyclopropane-1-carboxylic acid; ACO,aconitase; CAT, catalase; CAX, cation/proton exchanger; CS, cysteinesynthase; CSy, citrate synthase; ENO, enolase; FDH, formatedehydrogenase; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GR,glutathione reductase; Gly-I, glyoxalase I; GS, glutamine synthetase;GSH, reduced glutathione; LHC, light harvesting complex; LhcII-4,light-harvesting chlorophyll-a/b binding protein; LSU, large subunit; MTs,metallothioneins; MG, methylglyoxal; MDAR, monodehydroascorbatereductase; MDH, malate dehydrogenase; OEE, oxygen-evolving enhancerprotein; PC, Phytochelatin; Prx, peroxidoxin; PR, pathogenesis-related;PDH, pyruvate dehydrogenase; PSII-OEC 2, photosystem II oxygen-evolvingcomplex protein 2; PS, photosystem; ROS, reactive oxygen species;RuBisCO, ribulose-1,5-bisphosphate carboxylase oxygenase; SD, succinatedehydrogenase; SAM, S-adenosylmethionine; SSU, small subunit; Trx,thioredoxin; TPI, triosephosphate isomerase; TCA, tricarboxylic acid.

for vacuolar sequestration. Furthermore, increased abundanceof defense proteins for effective ROS scavenging and molecularchaperones for re-establishing normal protein conformation helpHM-stressed plants to maintain redox homeostasis. Modulationsof vital metabolic pathways – photosynthesis and mitochon-drial respiration – further help the stressed plant to producemore reducing power to compensate high-energy demand of HMchallenged cells.

Frontiers in Plant Science | Plant Proteomics January 2013 | Volume 3 | Article 310 | 2

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“fpls-03-00310” — 2013/1/24 — 17:14 — page 4 — #4


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pres

sion

/no

chan

gein

RO

Ssc

aven

ging

enzy

mes

.

Bon

aet

al.(

2007

)

O.s

ativ

aL.

cv.

Hw

ayeo

ng

(Ger

min

atin

gse

eds)

0.5

MTr

is–H

Cl(

pH8.

3),2

%

v/v

NP-

40,2

0m

MM

gCl 2

,2%

v/v

ME

,1m

MP

MS

F,1%

w/v

PV

P+

acet

one

9.5

Mur

ea,2

%v/

vN

P-40

,and

2.5%

v/v

phar

mal

ytes

(pH

3–10

:pH

5–8:

pH

4–6.

5=

1:3.

5:2.

5)

IEF

gel(

tube

gel),

2-D

E,

MA

LDI-T

OF

MS

25E

xces

sC

uin

duce

sox

idat

ive

stre

ssth

us

ham

perin

gm

etab

olic

proc

esse

s;up

-reg

ulat

ion

ofan

tioxi

dant

and

stre

ss-r

elat

edre

gula

tory

prot

eins

(gly

oxal

ase

I,pe

roxi

redo

xin)

help

to

mai

ntai

nce

llula

rho

meo

stas

is.

Ahs

anet

al.

(200

7b)

(Con

tinue

d)




“fpls-03-00310” — 2013/1/24 — 17:14 — page 5 — #5


Ta

ble

1|

Co

nti

nu

ed

Me

tal

Pla

nt

(tis

su

e)

Pro

tein

ex

tra

cti

on

bu

ffe

r+

pre

cip

ita

tio

n

Pro

tein

so

lub

iliz

ati

on

/

lysis

bu

ffe

r

Pro

teo

mic

me

tho

do

log

ies

IPM

ajo

rfi

nd

ing

sR

efe

ren

ce

BL.

albu

scv

.Rio

Mai

or(r

oot)

0.06

MD

TT,1

0%(w

/v)T

CA

in

cold

acet

one

with

0.06

MD

TT

2M

thio

urea

,7M

urea

,4%

(w/v

)CH

AP

S,0.

4%(v

/v)

Trito

nX-1

00,0

.06

MD

TT,a

nd

1%(v

/v)I

PG

buffe

r3–

10N

L

IPG

,2-D

E,

LC-M

S/M

S

128

Prot

eins

asso

ciat

edw

ithen

ergy

(gly

coly

sis,

TCA

cycl

e,ox

idat

ion–

redu

ctio

n),c

elld

ivis

ion,

prot

ein

met

abol

icpr

oces

ses

supp

ress

edun

der

Bde

ficie

ncy.

Alv

eset

al.(

2011

)

H.v

ulga

recv

s.

GP,

Cp,

Sh,

Cp

xS

h

DH

(Roo

t,le

af)

50m

Mph

osph

ate

buffe

r(p

H7.

5),

20m

MK

Cl,

0.5

MS

uc,1

0m

M

DTT

,0.2

mM

PM

SF,

10m

ME

DTA

,

10m

ME

GTA

+10

%(w

/v)T

CA

in

acet

one

0.5

MTE

AB

(pH

8.5)

cont

aini

ng0.

1%S

DS

iTR

AQ

pept

ide

tagg

ing,

MS

/MS

139

Hig

her

abun

danc

eof

Iron

defic

ienc

yse

nsiti

ve2

[IDS

2],I

DS

3,an

dm

ethy

lthio

-rib

ose

kina

se

obse

rved

inB

-tol

eran

tba

rley

islin

ked

to

side

roph

ore

prod

uctio

n

Patt

erso

net

al.

(200

7)

As

Ana

baen

asp

.

PC

C71

20(a

lgal

cells

)

10m

MTr

is–H

Cl(

pH8.

0),1

.5m

M

MgC

l 2,1

0m

MK

Cl+

10%

(w/v

)

TCA

inac

eton

e

7M

urea

,2M

thio

urea

,4%

CH

AP

S,40

mM

DTT

,and

1.0%

IPG

buffe

r(4

–7)

IPG

,2-D

E,

MA

LDI-T

OF,

and

LC-M

S

45U

p-re

gula

tions

ofP

GK

,FB

AII,

FBPa

se,T

K,A

TP

synt

hase

,Prx

,Trx

,oxi

dore

duct

ase

help

to

mai

ntai

nno

rmal

glyc

olys

is,P

PP,

and

turn

over

rate

ofC

alvi

ncy

cle,

prot

ectc

ells

from

oxid

ativ

e

stre

ss,t

here

byhe

lpin

gA

s-st

ress

accl

imat

ion.

Pand

eyet

al.

(201

2)

O.s

ativ

aL.

cv.

Don

gjin

(leaf

)

0.5

MTr

is–H

Cl(

pH8.

3),2

%(v

/v)

NP-

40,2

0m

MM

gCl 2

,2%

(v/v

)

ME

,1m

MP

MS

F,0.

7M

sucr

ose

+ac

eton

epr

ecip

itatio

n

8M

urea

,1%

CH

AP

S,0.

5%

(v/v

)IP

Gbu

ffer

pH4–

7,

20m

MD

TT

IPG

,2-D

E,

MA

LDI-T

OF

MS,

ES

I-MS

/MS

12E

nerg

yan

dm

etab

olis

mre

late

dpr

otei

nsov

er

expr

esse

din

dica

ting

high

eren

ergy

dem

and

unde

rAs

stre

ss;d

own-

regu

latio

nof

RuB

isC

O

and

chlo

ropl

ast

29kD

arib

onuc

leop

rote

ins

lead

tode

crea

sed

phot

osyn

thes

is.

Ahs

anet

al.

(201

0)

As

(V

and

III)

A.t

enui

s(le

af)

Gla

cial

acet

one

cont

aini

ng0.

07%

(v/v

)2-M

E,0

.34%

(w/v

)pla

nt

prot

ease

inhi

bito

r,an

d4%

(w/v

)

PV

P

4%(w

/v)C

HA

PS,

7M

urea

,

2M

thio

urea

,2%

(w/v

)DTT

,

1%(w

/v)p

harm

alyt

espH

3–10

,1%

(w/v

)res

olyt

espH

6–9.

5

IPG

,2-D

E,

MA

LDI-T

OF

MS

31A

str

eatm

ent

resu

lted

inpa

rtia

ldis

rupt

ion

of

the

phot

osyn

thet

icpr

oces

ses

with

prom

inen

t

frag

men

tatio

nof

the

Rub

isC

O.

Duq

uesn

oyet

al.

(200

9)

O.s

ativ

aL.

cv.

Don

gjin

(roo

t)

0.5

Mof

Tris

–HC

l(pH

8.3)

,2%

v/v

NP-

40,2

0m

MM

gCl 2

,2%

v/v

ME

,1m

MP

MS

F,0.

7M

sucr

ose

+ac

eton

epr

ecip

itatio

n

8M

urea

,1%

CH

AP

S,0.

5%

v/v

IPG

buffe

rpH

4–7,

20m

MD

TT

IPG

,2-D

E,

MA

LDI-T

OF

MS

23E

nerg

y,pr

imar

ym

etab

olic

path

way

s

supp

ress

edun

der

stre

ss;h

ighe

rG

SH

cont

ent

coup

led

with

enha

nced

expr

essi

ons

ofG

R,

SAM

S,G

STs,

CS,

GR

miti

gate

As-

indu

ced

oxid

ativ

est

ress

.

Ahs

anet

al.

(200

8)

Mn

V.un

guic

ulat

a[L

.]

Wal

p.cv

sTV

u91

,

TVu

1987

(leaf

)

700

mM

sucr

ose,

500

mM

Tris

,

50m

ME

DTA

,100

mM

KC

l,an

d

2%v/

vM

E+

wat

ersa

tura

ted

phen

ol

8M

urea

,2%

w/v

CH

AP

S,

0.5%

v/v

IPG

buffe

rpH

3–11

,

50m

MD

TT

IPG

,2-D

E,N

ano-

LC-M

S/M

S,E

SI

MS

/MS

8Lo

wer

abun

danc

eof

chlo

ropl

astic

prot

eins

invo

lved

inC

O2

fixat

ion

and

phot

osyn

thes

is

indi

cate

chan

neliz

ing

met

abol

icen

ergy

to

com

bat

the

Mn-

stre

ss;c

oord

inat

edin

terp

lay

of

apop

last

ican

dsy

mpl

astic

reac

tions

esse

ntia

l

for

stre

ssre

spon

se.

Führ

set

al.

(200

8) (Con

tinue

d)




“fpls-03-00310” — 2013/1/24 — 17:14 — page 6 — #6


Ta

ble

1|

Co

nti

nu

ed

Me

tal

Pla

nt

(tis

su

e)

Pro

tein

ex

tra

cti

on

bu

ffe

r+

pre

cip

ita

tio

n

Pro

tein

so

lub

iliz

ati

on

/

lysis

bu

ffe

r

Pro

teo

mic

me

tho

do

log

ies

IPM

ajo

rfi

nd

ing

sR

efe

ren

ce

Cr

M.s

inen

sis

cv.

Kosu

ng(r

oot)

0.5

MTr

is–H

Cl,

pH8.

3,2%

(v/v

)NP-

40,2

0m

MM

gCl 2

,

1m

MP

MS

F,2%

(v/v

)ME

,

and

1%(w

/v)P

VP

8M

urea

,1%

CH

AP

S,0.

5%

(v/v

)IP

Gbu

ffer

pH4–

7,

20m

MD

TT

IPG

,2-D

E,

MA

LDI-T

OF

MS,

MA

LDI-T

OF/

TOF

MS

36N

ovel

accu

mul

atio

nof

chro

miu

m-r

espo

nsiv

e

prot

eins

(e.g

.IM

Pase

,nitr

ate

redu

ctas

e,

aden

ine

phos

phor

ibos

yltr

ansf

eras

e,fo

rmat

e

dehy

drog

enas

e,pu

tativ

edi

hydr

olip

oam

ide

dehy

drog

enas

e)ob

serv

ed;C

rto

xici

tyis

linke

d

tohe

avy

met

alto

lera

nce

and

sene

scen

ce

path

way

s.

Sha

rmin

etal

.

(201

2)

P.su

bcap

itata

stra

in

Hin

dák

(alg

alce

lls)

500

mM

Tris

–HC

l(pH

8),

700

mM

sucr

ose,

10m

M

ED

TA,4

mM

asco

rbat

e,

0.4%

ME

,0.2

%Tr

iton

X-1

00

10%

,1m

MP

MS

F,1

μM

Leup

eptin

,0.1

mg/

mL

Pefa

bloc

+w

ater

satu

rate

d

phen

ol

7M

urea

,2M

thio

urea

,4%

CH

AP

S,50

mg/

mL

DTT

IPG

,2-D

E,

LC-E

SI-M

S/M

S

16C

r-str

ess

targ

etph

otos

ynth

etic

prot

eins

(RuB

isC

O,R

uBis

CO

activ

ase,

Ligh

tH

arve

stin

g

Chl

a/b

prot

ein

com

plex

,str

ess

rela

ted

Chl

a/b

bind

ing

prot

ein)

iden

tified

;Cr

also

indu

ces

mod

ulat

ion

ofpr

otei

nsin

volv

edin

amin

oac

ids

met

abol

ism

.

Vann

inie

tal.

(200

9)

Al

G.m

ax(L

.)M

err

cvs.

BaX

i10,

Ben

Di2

(roo

t)

10%

(w/v

)TC

Ain

acet

one

cont

aini

ng0.

07%

(w/v

)DTT

,

1%P

VP

7M

urea

,2M

thio

urea

,2%

(w/v

)CH

AP

S,1%

(w/v

)DTT

,

and

2%P

harm

alyt

epH

3–10

IPG

,2-D

E,

MA

LDI-T

OF

MS

30C

hape

rone

s,P

R10

,phy

toch

rom

eB

,

GTP

-bin

ding

prot

ein,

AB

Ctr

ansp

orte

r

ATP-

bind

ing

prot

ein

eith

erne

wly

indu

ced

or

up-r

egul

ated

,fac

ilita

test

ress

/def

ense

,sig

nal

tran

sduc

tion,

tran

spor

t,pr

otei

nfo

ldin

g,ge

ne

regu

latio

n,pr

imar

ym

etab

olis

ms.

Zhen

etal

.(20

07)

O.s

ativ

aL.

cv.

Xia

ngnu

o1

(XN

1)

(roo

t)

40m

MTr

is-b

ase,

5M

urea

,

2M

thio

urea

,2%

w/v

CH

AP

S,5%

w/v

PV

P,an

d

50m

MD

TT+

ice-

cold

acet

one

with

0.07

%(w

/v)

DTT

5M

urea

,2M

thio

urea

,4%

w/v

CH

AP

S,2%

v/v

IPG

buffe

r,40

mM

DTT

IPG

,2-D

E,

MA

LDI-T

OF/

TOF

MS,

MA

LDI-T

OF-

MS

17A

ntio

xida

tion

and

deto

xific

atio

nle

adby

up

regu

latio

nof

Al-r

espo

nsiv

epr

otei

ns(C

u–Zn

SO

D,G

ST,S

AM

S2)

,ulti

mat

ely

rela

ted

to

sulfu

rm

etab

olis

m.C

S,a

nove

lAl-i

nduc

ed

prot

ein,

play

key

role

inA

lres

ista

nce.

Yang

etal

.(20

07)

BA

BA

,β-a

min

obut

yric

acid

;C

S,cy

stei

nesy

ntha

se;

CH

AP

S,3-

[(3-c

hola

mid

opro

pyl)

dim

ethy

lam

mon

io]-1

-pro

pane

sulfo

nate

;C

p,C

lippe

r;FB

AII,

fruc

tose

bisp

hosp

hate

aldo

lase

II;FB

Pase

,fr

ucto

se1,

6bi

spho

s-ph

atas

e;G

P,G

olde

nPr

omis

e;IP

,num

ber

ofid

entifi

edpr

otei

ns;

PP

P,pe

ntos

eph

osph

ate

path

way

;Pr

x,pe

roxi

redo

xin;

PG

K,p

hosp

hogl

ycer

ate

kina

se;

SAM

S,S

-ade

nosy

lmet

hion

ine

synt

heta

se;

Sh,

Sah

ara;

TK,

tran

sket

olas

e;Tr

x,th

iore

doxi

n;TB

P,tr

ibut

ylph

osph

ine;

TEA

B,t

rieth

ylam

mon

ium

bica

rbon

ate;

TSP

P,ty

rosi

ne-s

peci

ficpr

otei

nph

osph

atas

epr

otei

ns;v

BP

O,v

anad

ium

-dep

ende

ntbr

omop

erox

idas

e.




“fpls-03-00310” — 2013/1/24 — 17:14 — page 7 — #7


COMPLEXATION, CHELATION, AND COMPARTMENTATION OF HMsWITHIN CELLOne of the important plant strategies of detoxifying HMs withincell is to synthesize low molecular weight chelators to minimizethe binding of metal ions to functionally important proteins (Ver-bruggen et al., 2009). The thiol-containing chelating compoundsstrongly interact with HM, thus reducing free HM ions fromcytosol and hence limiting HM toxicity (Cobbett and Golds-brough, 2002). The phytochelatins (PCs) and metallothioneins(MTs), the two best characterized cysteine-rich HM binding pro-tein molecules, play crucial roles in HM tolerance mechanism(Cobbett and Goldsbrough, 2002).

Phytochelatins synthesized from glutathione (GSH) by theenzyme PC synthase readily form complexes with HM in thecytosol and to facilitate their transport into vacuoles (Grill et al.,1989; Figure 1). Although PCs synthesis found to be inducedin presence of most of the studied HMs, modulation of pro-teins, amino acids involved in PC biosynthesis have been themost widely studied in response to Cd. Our recent compara-tive proteome analysis of high and low Cd accumulating soybeanshas revealed enhanced expression of glutamine synthetase (GS)under Cd stress. The enzyme GS is involved in the synthesis ofGSH through glutamate biosynthesis pathway (Sarry et al., 2006;Semane et al., 2010). The enhanced expression of GS leads to moreGSH formation (Hossain et al., 2012b). Induction of GSH syn-thesis implies higher metal binding capacity as well as enhancedcellular defense mechanism against oxidative stress (Verbruggenet al., 2009). Since GSH is the precursor of PC, enhanced expres-sion of GS helps the cell to synthesize and accumulate more PCto detoxify cytosolic Cd2+. Our finding is in agreement with pre-vious reports of up-regulation of GS in response to Cd (Kiefferet al., 2008; Hradilova et al., 2010; Semane et al., 2010; Ahsanet al., 2012). In contrast, sharp decline in GS abundance has beenreported in Cd-stressed rice roots (Lee et al., 2010).

Ahsan et al. (2012) exploited proteomic technique in combina-tion of metabolomics for deeper understanding of PC-mediateddetoxification of Cd2+ in soybean roots. Comparative analysisrevealed that proteins (GS beta 1) and amino acids (glycine, serine,glutamic acid) associated with Cd chelating pathways are highlyactive in low root-to-shoot Cd translocating cultivar. In addition,proteins involved in lignin biosynthesis were shown to be increasedunder stress. Proteomic findings suggest that translocation of Cdions from the root to the aerial parts might be prevented by theincreased xylem lignifications.

The PC biosynthetic pathway has been finely dissected in Cd-exposed Arabidopsis thaliana cells using protein and metaboliteprofiling (Sarry et al., 2006). At high Cd concentration globalpool of GSH decreased dramatically with the increase in dipeptideγGlu-Cys, suggesting high cellular demand of GSH for sustainingPC [(γGlu-Cys)n-Gly] synthesis.

Alvarez et al. (2009) implemented two quantitative proteomicsapproaches – 2-D DIGE and iTRAQ – to find out the relationbetween Cd2+ sequestration and thiol metabolism. Both tech-niques identified an increased abundance of proteins involved insulfur metabolism. Sulfite reductase and O-acetylserine sulfhydry-lase, involved in reduction of sulfate to cysteine, were found tobe overexpressed in Cd-treated Brassica juncea roots. Authors

suggested that under Cd-stress, sulfate availability for synthesisof PCs and GSH may limit Cd tolerance. Significant inductionsof GSH and PCs (PC3) in Cd-stressed rice roots further con-firm the role of thiol-peptides in HM tolerance mechanism (Ainaet al., 2007). Another proteomic study by Pandey et al. (2012)revealed higher abundance of cysteine synthase (CS) with highercontents of PCs and higher transcript of PC synthase in arsenicstressed Anabaena indicating their positive roles in As sequestra-tion. Arsenic induced increases in GSH and PCs were also recordedin fronds of arsenic hyperaccumulator Pteris vittata (Bona et al.,2011). Interestingly, no such increase was evident in roots underAs treatment. Proteomic results indicate that PCs could play rolein As detoxification in P. vittata fronds only, but overall PC medi-ated detoxification is not the primary mechanism of As-tolerancein As hyperaccumulator, but to other adaptive mechanism. Up-regulation of proteins (CS and GSTs) and GSH pool involved inAs detoxification has also been documented in proteomic study ofAs-stressed rice roots (Ahsan et al., 2008). Apart from Cd and Asstress, CS and GSH also play essential role in Al adaptation for rice(Yang et al., 2007) and soybean (Zhen et al., 2007).

Unlike PCs, proteomics-based report on HM-induced alter-ations of MTs is very limited. Zhang et al. (2009) for the first timeidentified MT-like proteins from Cu-stressed germinating rice seedembryo. A number of gene expression studies have shown that MTgenes are involved in Cu homeostasis and tolerance in Arabidop-sis (Murphy and Taiz, 1995) and Silene vulgaris (van Hoof et al.,2001). Plant MTs not only play vital role in chelating Cu throughthe Cys thiol groups but are also considered as a potent scavengerof ROS (Cobbett and Goldsbrough, 2002; Wang et al., 2004).

The final step of HM detoxification involves sequesteringof either free HMs or PCs-HMs complexes into cell vacuoles(Hall, 2002). This accumulation is mediated by tonoplast-boundcation/proton exchanger, P-type ATPase and ATP-dependent ABCtransporter (Salt and Rauser, 1995; Hall, 2002). Transportersare also situated in plasma membrane and facilitate transportof HMs into apoplast. As the vacuoles or apoplasts have limitedmetabolic activity, accumulations of HMs in these compartmentsreduce the toxic effects of HMs (Schneider et al., 2009). TheiTRAQ analysis of Cd-exposed barley leaf mesophyll tonoplastproteome led to the identification of ∼50 vacuolar transporters,that include vacuolar ATPase subunits, MRP-like ABC transporterand two novel CAX transporters (CAX1a and CAX5) and oneAl-activated malate transporter protein (Schneider et al., 2009).Induction of these transporters especially cation/proton exchanger1a and ABC transporter assure Cd2+ transport into vacuole(Aina et al., 2007). Further proteomic study by Lee et al. (2010)revealed induction of vacuolar proton-ATPase in rice roots andleaves indicating their positive role in Cd detoxification throughvacuolarisation.

HM-INDUCED OXIDATIVE STRESS AND ALTERATION OF REDOXHOMEOSTASISCellular ROS generation gets accelerated upon exposure to HMstress. HMs (Cu, Fe, Cr) that are directly involved in cellular redoxreaction lead to ROS generation known as redox active, while redoxinactive HMs (Cd, Al, As, Ni) trigger oxidative stress by depletingcells major thiol-containing antioxidants and enzymes, disrupting




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electron transport chain or by inducing lipid peroxidation (Ercalet al., 2001; Hossain et al., 2012a). The excess intracellular ROSlevel alters protein structure by inducing oxidation of both proteinbackbone and amino acid side chain residues (Villiers et al., 2011).To counter stress, plants have evolved robust antioxidant defensemechanism comprised of both enzymatic and non-enzymaticcomponents (Hossain et al., 2012d).

Most of the proteomic research done so far on HM-related tox-icity revealed positive correlation between tolerance and increasedabundance of scavenger proteins. Within plant cells, SOD con-stitutes the first line of defense against ROS. It plays pivotalrole in cellular defense against oxidative stress, as its activ-ity directly modulates the amount of O•−

2 and H2O2, the twoimportant Haber–Weiss reaction substrates. The excess O•−

2 gen-erated under HM-stress usually disproportionate into H2O2 bythe action of SOD, which is then metabolized by the componentsof the ascorbate–GSH cycle. Higher expressions of SOD isoforms(Cu/Zn-SOD, Fe-SOD) have been documented in plants exposedto excess Cd (Kieffer et al., 2008, 2009; Alvarez et al., 2009; Far-inati et al., 2009; Semane et al., 2010; Hossain et al., 2012b) andAl (Yang et al., 2007). Interestingly, root proteome analysis of Cd-exposed B. juncea revealed up-regulation of Fe-SOD while downregulation of Cu/Zn SOD (Alvarez et al., 2009). Ascorbate peroxi-dase (APX), peroxidase (POD), and catalase (CAT) are involved inscavenging H2O2, hence protecting cell membrane from hydroxylradical-induced lipid peroxidation (Barber and Thomas, 1978).The scavenging roles of APX, POD, and CAT have been docu-mented in several proteomic studies related to Cd stress (Sarryet al., 2006; Aina et al., 2007; Kieffer et al., 2008; Lee et al., 2010;Hossain et al., 2012b) and As (Pandey et al., 2012) toxicity. Inter-estingly, excessive Cu (Bona et al., 2007), Cr (Sharmin et al., 2012)treatments or B deficiency (Alves et al., 2011) lead to decreasedabundance of APX and POD. The detected suppression of POD isin accordance with the decrease in POD reported in maize rootstreated with Al (Wang et al., 2011).

The abundance of another antioxidant enzyme of ascorbate–GSH cycle, monodehydroascorbate reductase (MDAR) was foundto be increased in response to Cd (Sarry et al., 2006; Alvarez et al.,2009). MDAR helps to scavenge monodehydroascorbate radi-cal and by doing this it generates dehydroascorbate (DHA), theoxidized form of ascorbate. Up-regulation of MDAR assures pro-duction of DHA, the substrate of dehydroascorbate reductase(DHAR) enzyme that catalyzes reduction of DHA to AsA (reducedascorbate). In contrary, shoot proteome analysis of Arabidopsishalleri has shown decreased expression of MDAR in response toCd, Zn, and rhizosphere microorganisms (Farinati et al., 2009).This down-regulation is also evident in roots of Lupinus albusundergoing long-term B deficiency (Alves et al., 2011). DecreasedMDAR abundance in HM-stressed plants might indicate non-enzymatic disproportionation of monodehydroascorbate intoAsA, essential for maintenance of balanced redox status (Hossainet al., 2009). Yet another well documented antioxidant found to beup-regulated under HM stress is peroxiredoxin (Prx). The Prx isbasically a thiol peroxidase with multiple functions. It (a) detox-ifies hydroperoxides; (b) plays essential role in enzyme activationand redox sensoring; (c) acts as molecular chaperone similar toHSPs; (d) induces cell signaling (Dietz, 2003; Dietz et al., 2006;

Jang et al., 2004; Barranco-Medina et al., 2009). Prx was found tobe induced under Cd (Sarry et al., 2006; Ahsan et al., 2007a; Hos-sain et al., 2012b) and As (Requejo and Tena, 2006; Pandey et al.,2012) stress.

Plants are also equipped with some additional defense pro-teins, shown to be up-regulated by HM stress. This group includesthioredoxin (Trx), Trx-dependent peroxidase, NADP(H)-oxido-reductase and glyoxylase I (Gly I). Trx is known to suppressapoptosis as well as supplies reducing equivalents to antioxidants(Hishiya et al., 2008). Excess Cu treatment seems to down-regulatethe abundance of Trx and Trx-POD in germinating rice embryo(Zhang et al., 2009) and Cannabis sativa roots (Bona et al., 2007)respectively. However, enhanced expression of Trx was found tobe helpful in mitigating oxidative stress in As-treated Anabaena(Pandey et al., 2012).

Methylglyoxal (MG), a cytotoxic by-product of glycolysis gen-erally accumulated in cell in response to environmental stressesincluding HM (Espartero et al., 1995). MG readily interacts withnucleic acids and proteins causing alteration of function (Yadavet al., 2005). Detoxification of MG through glyoxalase pathwayinvolves active participation of GSH and Gly I and Gly II enzymes.Up-regulation of Gly I was found to help the germinating riceseedlings in detoxifying MG under Cd (Ahsan et al., 2007a) andCu (Ahsan et al., 2007b). Higher Gly I abundance was also reportedin Cd + Zn + microorganisms treated A. halleri shoots (Farinatiet al., 2009). Proteomic study also highlighted enhanced expres-sion of NADP(H)-oxido-reductase by Cd (Sarry et al., 2006; Leeet al., 2010) and As (Pandey et al., 2012). This protein is a vitalcomponent of plants second line of defense, protecting cells fromHM-induced oxidative damages.

Plants tolerance against HMs is often attributed to steady stateof GSH pool for its multifunctional activities in PC synthesis,MG detoxification, ROS scavenging through ascorbate–GSH cycle,GSTs mediated decomposition of toxic compounds as well as stresssignaling (Figure 1). Within GSH cycle, glutathione reductase(GR) acts as a rate limiting enzyme that catalyzes reduction of oxi-dized glutathione (GSSG) to GSH (reduced glutathione) and withthe help of DHAR it maintains high AsA/DHA ratio necessary fortight control of HM-induced ROS scavenging. The delicate balancebetween GSH and GSSG is critical for keeping a favorable redoxstatus for the detoxification of H2O2. Higher abundance of GSTshas been observed in response to Cd (Alvarez et al., 2009; Lee et al.,2010), As (Ahsan et al., 2008; Pandey et al., 2012), Cu (Zhang et al.,2009). Findings of Ahsan et al. (2008) revealed increased activityof GST-omega in rice roots following exposure of AsV, indicatingthe probable role of GST-omega in inorganic arsenic biotransfor-mation and metabolism. The authors also suggested that depletionin GSH content may be associated with high rate of PCs synthesisthus detoxification of As through compartmentalization or due todown-regulation of enzymes of GSH biosynthetic pathways suchas GR and CS. The HM-induced PCs synthesis coupled with GSHdepletion is in agreement with earlier studies by Sarry et al. (2006)and Di Baccio et al. (2005).

Proteomic analyses strongly indicate that accumulation ofdefense proteins chiefly enzymatic components of ascorbate–GSHcycle, POD, CAT, GSH, GSTs, Gly I, Prx, Trx help cells to mitigateHM-induced oxidative stress by scavenging ROS.




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MOLECULAR CHAPERONESProtein dysfunction is an inevitable consequence of a wide range ofadverse environmental conditions including HM toxicity. Molec-ular chaperones/heat-shock proteins (HSPs) are responsible forprotein stabilization, proper folding, assembly, and transloca-tion under both optimum and adverse growth conditions (Wanget al., 2004). In our study, enhanced abundance (>2-fold) ofHSP70 protein was detected in leaves of high Cd-accumulatingsoybean cultivar Harosoy while low Cd-accumulating cv. Fukuyu-taka exhibited decreased expression (Hossain et al., 2012b). Cd-induced up-regulation of HSP70 is also evident in response tovarious HMs including Cd (Kieffer et al., 2009; Hradilova et al.,2010; Rodríguez-Celma et al., 2010), Cr (Sharmin et al., 2012),and B deficiency (Alves et al., 2011). Ahsan et al. (2007a) reportedincrease of DnaK-type molecular chaperone BiP and chaperoneprotein HchA in germinating rice seedlings exposed to acute Cdtoxicity. Al-stress also is known to induce one LMW-HSP andthree DnaJ-like proteins in Al-stressed soybean (Zhen et al., 2007).To sum up, HSPs/chaperones play pivotal role in combating HMstress by re-establishing normal protein conformation and hence,cellular homeostasis.

HM-INDUCED ALTERATION OF PROTEINS INVOLVED INPHOTOSYNTHESIS AND ENERGY METABOLISMDown-regulation of photosynthetic machinery is a known phe-nomenon of Cd stress. Low abundance of proteins involved inphotosynthetic electron transport chain and Calvin cycle has beenreported in Cd-exposed Populus (Kieffer et al., 2008, 2009; Durandet al., 2010) and Thlaspi (Tuomainen et al., 2006). Pioneer pro-teomic work by Hajduch et al. (2001) of rice leaves exposed toHMs revealed drastic reduction in abundance/fragmentation oflarge and small subunits of RuBisCO (LSU and SSU), suggest-ing complete disruption of photosynthetic machinery by HMstress. This decrease in RuBisCO has also been documentedin other HMs toxicity like As (Duquesnoy et al., 2009) and Cd(Kieffer et al., 2008). Proteomic analysis for other toxic HMslike As-exposed leaf proteome of Agrostis tenuis has showntotal disruption of RuBisCO LSU and SSU along with oxygen-evolving enhancer protein 1 and oxygen evolving protein 2 inresponse to 134 μM As(V) treatment (Duquesnoy et al., 2009).Potassium dichromate treatment had similar effects on algalRuBisCO LSU and some antenna proteins namely light harvest-ing Chl a/b protein complex. However, Vannini et al. (2009)reported higher abundance of RuBisCO activase in Pseudokirch-neriella subcapitata under chromate treatment. Interestingly, inour proteomic experiment with Cd-exposed soybean, increasedabundance of RuBisCO LSU-binding protein subunits alphaand beta, RuBisCO activase, oxygen-evolving enhancer protein1 and 2, NAD(P)H-dependent oxido-reductase, photosystemI and II-related proteins were evident (Hossain et al., 2012b).Enhanced expressions of proteins involved in photosystem I,II, and Calvin cycle might be an adaptive feature to over-come the Cd injury in soybean. This increased abundance is inaccordance with the findings of Semane et al. (2010), who alsoreported increase of photosynthetic protein abundance in leavesof Arabidopsis treated with mild Cd stress. In our opinion, contri-bution of high photosynthetic assimilates into respiration would

help plants to yield more energy needed to combat the Cd2+stress.

To maintain the normal growth and development understressed environment, plants need to up regulate metabolicpathways such as glycolysis and tricarboxylic acid (TCA) cycle.Detailed analysis of HM toxicity-related proteomic works hasshown higher abundance of glycolytic enzymes phosphoglycer-ate mutase (PGM), glucose-6-phosphate isomerase (G6PI), triosephosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydro-genase (G3PDH), enolase (ENO), and pyruvate kinase (PK) inresponse to Cd (Sarry et al., 2006; Kieffer et al., 2008; Rodríguez-Celma et al., 2010; Hossain et al., 2012b), Cr (Labra et al., 2006).However, down-regulation of G3PDH was reported in As-treatedrice roots (Ahsan et al., 2008) and roots of Lupinus albus under Bdeficiency (Alves et al., 2011). Similarly, Cu-treated Cannabis rootsexhibited down-regulation of another glycolytic enzyme ENO,the metalloenzyme that catalyzes penultimate step of glycolysis –conversion of 2-phosphoglycerate to phosphoenolpyruvate (Bonaet al., 2007).

Like glycolysis, enzymes of TCA cycle citrate synthase (CS),succinate dehydrogenase (SD), malate dehydrogenase (MDH),aconitase (ACO), aconitate hydratase (AH) were found to be up-regulated under Cd stress (Sarry et al., 2006; Kieffer et al., 2009;Rodríguez-Celma et al., 2010; Semane et al., 2010; Hossain et al.,2012b; Figure 1). In contrast, suppressions of several AH isoformswere evident in long-term B deficiency (Alves et al., 2011). Overall,up-regulation of glycolysis and TCA cycle might help the stressedplant to produce more reducing power to compensate high-energydemand of HM challenged cell.

ACCUMULATION OF PR PROTEINS IN RESPONSE TO HM STRESSPlant cells trigger some common defense machineries wheneverthey encounter a biotic or abiotic stress. Accumulation of PRproteins is one of such plant defense strategies and often asso-ciated with systemic acquired resistance (SAR) against a widerange of pathogens (Van Loon, 1997; Durrant and Dong, 2004).Using the 2-DE approach, Elvira et al. (2008) successfully iden-tified different PR protein isoforms (viz. PR-1, β-1,3-glucanasesPR-2, chitinases PR-3, osmotin-like protein PR-5, peroxidases PR-9, germin-like protein PR-16, and NtPRp27-like protein PR-17) inCapsicum chinense leaves and additionally resolved their specificaccumulation pattern in both the compatible and incompatiblePMMoV–C. chinense interactions. Apart from the assigned rolein plant defense against pathogenic constraints, PR proteins alsoplay key role in adaptation to stressful environments includingHM toxicity (Hensel et al., 1999; Rakwal et al., 1999; Van Loonand Van Strien, 1999; Hajduch et al., 2001; Akiyama et al., 2004;Edreva, 2005). Kieffer et al. (2008) documented marked increase inabundance of PR proteins class I chitinases (PR-3 family), severalβ-1,3-glucanases (PR-2 family), and thaumatin-like protein (PR-5family) in Cd-exposed poplar leaves. Endo-1,3-beta-glucanase, aclass 2 PR protein, also found to be induced in rice roots undershort-term Cd stress (Lee et al., 2010). Higher abundance of PRproteins under HM as documented in many proteomic studies isin accordance with previous transcriptomic analysis of mercuricchloride-treated Zea mays leaves (Didierjean et al., 1996). Like Cdstress, PR-10 and LIR18B protein (both belong to PR-10 family),




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and an acidic chitinase (PR-8 family) were de novo expressed underB deficiency (Alves et al., 2011). Stress-induced increase in ROSlevel has been shown to induce PR protein accumulation (Jwaet al., 2006). Treatment with excess Cu increased abundance oftwo PR proteins (PR-10a and putative PR proteins) in germinatingrice embryos (Zhang et al., 2009). Analysis of the Vigna unguic-ulata leaf apoplast proteome using 2-DE and LC-MS/MS alsorevealed accumulation of several PR-like proteins glucanase, chiti-nase, and thaumatin-like proteins in response to excess Mn supply(Fecht-Christoffers et al., 2003). Transgenic tobacco overexpress-ing pepper gene CABPR1 encoding basic PR-1 protein showedenhanced resistance against HMs as well as pathogen stresses(Sarowar et al., 2005). These transgenic lines exhibited significantdecline in total POD activity, suggesting that overexpression ofCABPR1 in tobacco cells altered redox balance. Although, the pre-cise role of PR proteins in combating HM stress is not yet clearlyunderstood, the authors suggested that the induced redox imbal-ance might lead to H2O2 accumulation, triggering stress tolerancecascade. Several in vitro experiments have demonstrated that PRproteins display additional functions related to growth and devel-opment by modulating signal molecules (Kasprzewska, 2003; Liuand Ekramoddoullah, 2006). However, further proteomic investi-gations need to be undertaken to resolve the underlying molecularmechanism of PR proteins mediated plants HM tolerance.

CONCLUSION AND FUTURE PROSPECTSThe present review outlines the impact of HMs stresses onplant proteome constituents. Most of the investigations done sofar primarily highlighted the differential expression of proteinsinvolved in plant defense and detoxification pathways, namely ROSscavenging, chelation, compartmentalization. In addition, accu-mulation of PR proteins and modulation of plants vital metabolicpathways CO2 assimilation, mitochondrial respiration in main-taining steady state of reducing power and energy required forcombating HM-induced stress has been discussed in detail. Care-ful analysis of published proteomic works on HM toxicity hasrevealed that classical 2-DE coupled with MS-based protein iden-tification has been the most widely used proteomic technique ininvestigating plant HM tolerance at organ/whole plant level. Theseproteomic findings have enriched us for deeper understandingplants HM tolerance mechanism.

The cellular mechanism of sensing stress and transduction ofstress signals into the cell organelle represent the initial reaction ofplant cells toward any kind of stress including HM. Communica-tion through intracellular compartments plays a significant role in

stress signal transduction process that finally activates defense genecascade (Hossain et al., 2012d). To dissect the underlying molecu-lar mechanism of how a plant cell modulates its protein signatureto cope with stress, in depth study on organelle proteome would beof great contribution toward development of HM-tolerant crops.

As the PCs mediated HM-ion detoxification pathway ends insequestration of PC-HM complexes into vacuole through vari-ous transporter proteins present in tonoplast membrane, moreresearch on vacuole proteome needs to be undertaken for identi-fication and characterization of novel metal transporter proteinsresponsible for cytoplasmic efflux of transition metal cations intovacuole. Legendary work by Schneider et al. (2009) on quanti-tative detection of changes in barley leaf mesophyll tonoplastproteome using advanced gel free iTRAQ method has enriched ourknowledge about contribution of vacuolar transporters to Cd2+detoxification. Plasma membrane proteome should be anothertarget of future proteomic research on HM stress, as it acts as a pri-mary interface between the cellular cytoplasm and the extracellularenvironment, thus playing a vital role in stress signal perceptionand transduction. Furthermore, transporter proteins present incell membrane have importance in up-taking HM-ions into thecell. As most of the organelle membrane proteins are hydrophobicin nature, MS-based gel free system would be the most promisingtechnique for identification of such proteins.

Plants response to multiple HMs would be another interestingarea of future proteomic research (Sharma and Dietz, 2009). Thiscould shed some light on cross talk between different HM stresssignal pathways.

Heavy metal-induced protein oxidation study through redoxproteomic approach has been the focus of much interest. More ini-tiatives in this topic need to be taken as PTM/redox modificationof proteins provides fundamental information about HM toxic-ity mechanism and biomarker discovery (Dowling and Sheehan,2006; Braconi et al., 2011).

In summary, we believe that more research on sub-proteome-based HM approach would provide new insights into plantsHM-stress response mechanism. HM-induced novel marker pro-teins would further enable us to design HM-tolerant transgeniccrops.

ACKNOWLEDGMENTSThe authors thankfully acknowledge support from the Depart-ment of Science and Technology, Government of India, throughDST-BOYSCAST Fellowship Programme and National Agricul-ture and Food Research Organization, Japan.

REFERENCESAhsan, N., Nakamura, T., and Komatsu,

S. (2012). Differential responses ofmicrosomal proteins and metabo-lites in two contrasting cadmium(Cd)-accumulating soybean cultivarsunder Cd stress. Amino Acids 42,317–327.

Ahsan, N., Lee, D. G., Alam, I.,Kim, P. J., Lee, J. J., Ahn, Y. O.,et al. (2008). Comparative proteomicstudy of arsenic-induced differen-tially expressed proteins in rice roots

reveals glutathione plays a centralrole during As stress. Proteomics 8,3561–3576.

Ahsan, N., Lee, D. G., Kim, K. H., Alam,I., Lee, S. H., Lee, K. W., et al. (2010).Analysis of arsenic stress-induced dif-ferentially expressed proteins in riceleaves by two-dimensional gel elec-trophoresis coupled with mass spec-trometry. Chemosphere 78, 224–231.

Ahsan, N., Lee, D. G., Lee, S. H., Kang, K.Y., Lee, J. J., Kim, P. J., et al. (2007b).Excess copper induced physiological

and proteomic changes in germi-nating rice seeds. Chemosphere 67,1182–1193.

Ahsan, N., Lee, S. H., Lee, D. G.,Lee, H., Lee, S. W., Bahk, J. D.,et al. (2007a). Physiological and pro-tein profiles alternation of germinat-ing rice seedlings exposed to acutecadmium toxicity. C. R. Biol. 330,735–746.

Ahsan, N., Renaut, J., and Komatsu,S. (2009). Recent developments inthe application of proteomics to the

analysis of plant responses to heavymetals. Proteomics 9, 2602–2621.

Aina, R., Labra, M., Fumagalli, P.,Vannini, C., Marsoni, M., Cucchi,U., et al. (2007). Thiol-peptide leveland proteomic changes in response tocadmium toxicity in Oryza sativa L.roots. Environ. Exp. Bot. 59, 381–392.

Akiyama, T., Pillai, M. A., and SentokuN. (2004). Cloning, characterizationand expression of OsGLN2, a riceendo-1,3-beta-glucanase gene regu-lated developmentally in flowers and




“fpls-03-00310” — 2013/1/24 — 17:14 — page 11 — #11


hormonally in germinating seeds.Planta 220, 129–139.

Alvarez, S., Berla, B. M., Sheffield,J., Cahoon, R. E., Jez, J. M., andHicks, L. M. (2009). Comprehen-sive analysis of the Brassica juncearoot proteome in response to cad-mium exposure by complementaryproteomic approaches. Proteomics 9,2419–2431.

Alves, M., Moes, S., Jenö, P., Pinheiro,C., Passarinho, J., and Ricardo, C. P.(2011). The analysis of Lupinus albusroot proteome revealed cytoskele-ton altered features due to long-termboron deficiency. J. Proteomics 74,1351–1363.

Barber, D. J. W., and Thomas, J. K.(1978). Reactions of radicals withlecithin bilayers. Radiat. Res. 74,51–65.

Barranco-Medina, S., Lázaro, J. J., andDietz, K. J. (2009). The oligomericconformation of peroxiredoxins linksredox state to function. FEBS Lett.583, 1809–1816.

Bohnert, H. J., Gong, Q., Li, P., andMa, S. (2006). Unraveling abioticstress tolerance mechanisms – gettinggenomics going. Curr. Opin. PlantBiol. 9, 180–188.

Bona, E., Marsano, F., Massa, N., Cat-taneo, C., Cesaro, P., Argese, E.,et al. (2011). Proteomic analysis as atool for investigating arsenic stress inPteris vittata roots colonized or notby arbuscular mycorrhizal symbiosis.J. Proteomics 74, 1338–1350.

Bona, E., Marsano, F., Cavaletto, M., andBerta, G. (2007). Proteomic charac-terization of copper stress responsein Cannabis sativa roots. Proteomics7, 1121–1130.

Braconi, D., Bernardini, G., and San-tucci, A. (2011). Linking proteinoxidation to environmental pollu-tants: redox proteomic approaches. J.Proteomics 74, 2324–2337.

Bubb, J. M., and Lester, J. N. (1991).The impact of heavy metals on low-land rivers and the implications forman and the environment. Sci. TotalEnviron. 100, 207–233.

Cobbett, C. S. (2000). Phytochelatinsand their roles in heavy metal detoxi-fication. Plant Physiol. 123, 825–832.

Cobbett, C., and Goldsbrough, P.(2002). Phytochelatins and metal-lothioneins: roles in heavy metaldetoxification and homeostasis.Annu. Rev. Plant Biol. 53, 159–182.

Di Baccio, D., Kopriva, S., Sebastiani,L., and Rennenberg, H. (2005). Doesglutathione metabolism have a rolein the defence of poplar against zincexcess? New Phytol. 167, 73–80.

Didierjean, L., Frendo, P., Nasser, W.,Genot, G., Marivet, J., and Burkard,

G. (1996). Heavy-metal-responsivegenes in maize: identification andcomparison of their expression uponvarious forms of abiotic stress. Planta199, 1–8.

Dietz, K. J. (2003). Plant peroxiredoxins.Annu. Rev. Plant Biol. 54, 93–107.

Dietz, K. J., Jacob, S., Oelze, M. L.,Laxa, M., Tognetti, V., de Miranda,S. M., et al. (2006). The functionof peroxiredoxins in plant organelleredox metabolism. J. Exp. Bot. 57,1697–1709.

Dowling, V. A., and Sheehan, D. (2006).Proteomics as a route to identifi-cation of toxicity targets in envi-ronmental toxicology. Proteomics 6,5597–5604.

Durand, T. C., Sergeant, K., Planchon,S., Carpin, S., Label, P., Mora-bito, D., et al. (2010). Acute metalstress in Populus tremula × P. alba(717-1B4 genotype): leaf and cambialproteome changes induced by cad-mium(2+). Proteomics 10, 349–368.

Durrant, W. E., and Dong, X. (2004).Systemic acquired resistance. Annu.Rev. Phytopathol. 42, 185–209.

Duquesnoy, I., Goupil, P., Nadaud, I.,Branlard, G., Piquet-Pissaloux, A.,and Ledoigt, G. (2009). Identifica-tion of Agrostis tenuis leaf proteinsin response to As(V) and As(III)induced stress using a proteomicsapproach. Plant Sci. 176, 206–213.

Edreva, A. (2005). Pathogenesis-relatedproteins. Research progress in the last15 years. Gen. Appl. Plant Physiol. 31,105–124.

Elvira, M. I., Galdeano, M. M., Gilardi,P., García-Luque, I., and Serra, M.T. (2008). Proteomic analysis ofpathogenesis-related proteins (PRs)induced by compatible and incom-patible interactions of pepper mildmottle virus (PMMoV) in Capsicumchinense L3 plants. J. Exp. Bot. 59,1253–1265.

Ercal, N., Gurer-Orhan, H., and Aykin-Burns, N. (2001). Toxic metals andoxidative stress part I: mechanismsinvolved in metal-induced oxidativedamage. Curr. Top. Med. Chem. 1,529–539.

Espartero, J., Sanchez-Aguayo, I., andPardo, J. M. (1995). Molecular char-acterization of glyoxalase-I from ahigher plant; upregulation by stress.Plant Mol. Biol. 29, 1223–1233.

Farinati, S., DalCorso, G., Bona, E., Cor-bella, M., Lampis, S., Cecconi, D.,et al. (2009). Proteomic analysis ofArabidopsis halleri shoots in responseto the heavy metals cadmium andzinc and rhizosphere microorgan-isms. Proteomics 9, 4837–4850.

Fecht-Christoffers, M. M., Braun, H. P.,Lemaitre-Guillier, C., VanDorsselaer,

A., and Horst, W. J. (2003). Effect ofmanganese toxicity on the proteomeof the leaf apoplast in cowpea. PlantPhysiol. 133, 1935–1946.

Führs, H., Hartwig, M., Molina,L. E., Heintz, D., Van Dorsselaer,A., Braun, H. P., et al. (2008).Early manganese-toxicity response inVigna unguiculata L. – a proteomicand transcriptomic study. Proteomics8, 149–159.

Grill, E., Loffler, S., Winnacker, E.L., and Zenk, M. H. (1989). Phy-tochelatins, the heavy-metal-bindingpeptides of plants, are synthe-sized from glutathione by a specificgamma-glutamylcysteine dipeptidyltranspeptidase (phytochelatin syn-thase). Proc. Natl. Acad. Sci. U.S.A.86, 6838–6842.

Gygi, S. P., Rochon, Y., Franza, B. R.,and Aebersold, R. (1999). Correlationbetween protein and mRNA abun-dance in yeast. Mol. Cell. Biol. 19,1720–1730.

Hajduch, M., Rakwal, R., Agrawal,G. K., Yonekura, M., and Pre-tova, A. (2001). High-resolutiontwo-dimensional electrophoresisseparation of proteins from metal-stressed rice (Oryza sativa L.) leaves:drastic reductions/fragmentation ofribulose-1,5-bisphosphate carboxy-lase/oxygenase and induction ofstress-related proteins. Electro-phoresis 22, 2824–2831.

Hall, J. L. (2002). Cellular mechanismsfor heavy metal detoxification andtolerance. J. Exp. Bot. 53, 1–11.

Hensel, G., Kunze, G., and Kunze, I.(1999). Expression of the tobaccogene CBP20 in response to devel-opmental stage, wounding, salicylicacid and heavy metals. Plant Sci. 148,165–174.

Hishiya, S., Hatakeyama, W., Mizota,Y., Hosoya-Matsuda, N., Motohashi,K., Ikeuchi, M., et al. (2008). Binaryreducing equivalent pathways usingNADPH-thioredoxin reductase andferredoxin-thioredoxin reductase inthe cyanobacterium Synechocystis sp.strain PCC 6803. Plant Cell Physiol.49, 11–18.

Hossain, M. A., Piyatida, P., Teixeira daSilva, J. A., and Fujita, M. (2012a).Molecular mechanism of heavy metaltoxicity and tolerance in plants: cen-tral role of glutathione in detoxifica-tion of reactive oxygen species andmethylglyoxal and in heavy metalchelation. J. Bot. 2012, 1–37.

Hossain, Z., Hajika, M., and Komatsu,S. (2012b). Comparative proteomeanalysis of high and low cadmiumaccumulating soybeans under cad-mium stress. Amino Acids 43, 2393–2416.

Hossain, Z., Lopez-Climent, M. F.,Arbona, V., Perez-Clemente, R.M., and Gomez-Cadenas, A. (2009).Modulation of the antioxidant sys-tem in citrus under waterlogging andsubsequent drainage. J. Plant Physiol.166, 1391–1404.

Hossain, Z., Makino, T., and Komatsu,S. (2012c). Proteomic study ofβ-aminobutyric acid-mediated cad-mium stress alleviation in soybean. J.Proteomics 75, 4151–4164.

Hossain, Z., Nouri, M. Z., and Komatsu,S. (2012d). Plant cell organelle pro-teomics in response to abiotic stress.J. Proteome Res. 11, 37–48.

Hradilova, J., Rehulka, P., Rehulkova,H., Vrbova, M., Griga, M., andBrzobohaty, B. (2010). Compara-tive analysis of proteomic changes incontrasting flax cultivars upon cad-mium exposure. Electrophoresis 31,421–431.

Jang, H. H., Lee, K. O., Chi, Y. H., Jung,B. G., Park, S. K., Park, J. H., et al.(2004). Two enzymes in one; twoyeast peroxiredoxins display oxidativestress-dependent switching from aperoxidase to a molecular chaperonefunction. Cell 117, 625–635.

Jwa, N. S., Agrawal, G. K., Tamogami,S., Yonekura, M., Han, O., Iwa-hashi, H., et al. (2006). Role ofdefense/stress related marker genes,proteins and secondary metabolitesin defining rice self-defense mecha-nisms. Plant Physiol. Biochem. 44,261–273.

Kieffer, P., Dommes, J., Hoffmann,L., Hausman, J. F., and Renaut, J.(2008). Quantitative changes in pro-tein expression of cadmium-exposedpoplar plants. Proteomics 8, 2514–2530.

Kieffer, P., Planchon, S., Oufir, M.,Ziebel, J., Dommes, J., Hoffmann,L., et al. (2009). Combining pro-teomics and metabolite analyses tounravel cadmium stress-response inpoplar leaves. J. Proteome Res. 8,400–417.

Komatsu, S., and Ahsan, N. (2009). Soy-bean proteomics and its applicationto functional analysis. J. Proteomics72, 325–336.

Kasprzewska, A. (2003). Plant chitinases– regulation and function. Cell. Mol.Biol. Lett. 8, 809–824.

Labra, M., Gianazza, E., Waitt, R.,Eberini, I., Sozzi, A., Regondi, S.,et al. (2006). Zea mays L. proteinchanges in response to potassiumdichromate treatments. Chemosphere62, 1234–1244.

Lee, K., Bae, D. W., Kim, S. H., Han,H. J., Liu, X., Park, H. C., et al.(2010). Comparative proteomic anal-ysis of the short-term responses of




“fpls-03-00310” — 2013/1/24 — 17:14 — page 12 — #12


rice roots and leaves to cadmium. J.Plant Physiol. 167, 161–168.

Liu, J. J., and Ekramoddoullah, A.(2006). The family 10 of plantpathogenesis-related proteins: theirstructure, regulation, and function inresponse to biotic and abiotic stresses.Physiol. Mol. Plant Pathol. 68, 3–13.

Luque-Garcia, J. L., Cabezas-Sanchez,P., and Camara, C. (2011). Pro-teomics as a tool for examining thetoxicity of heavy metals. Trends Anal.Chem. 30, 703–716.

Ma, J. F., Ryan, P. R., and Delhaize,E. (2001). Aluminium tolerance inplants and the complexing role oforganic acids. Trends Plant Sci. 6,273–278.

Maksymiec, W. (2007). Signalingresponses in plants to heavy metalstress. Acta Physiol. Plant. 29,177–187.

Murphy, A., and Taiz, L. (1995).Comparison of metallothionein geneexpression and nonprotein thiols inten Arabidopsis ecotypes. Correlationwith copper tolerance. Plant Physiol.109, 945–954.

Pandey, S., Rai, R., and Rai, L. C. (2012).Proteomics combines morphologi-cal, physiological and biochemicalattributes to unravel the survivalstrategy of Anabaena sp. PCC7120under arsenic stress. J. Proteomics 75,921–937.

Patterson, J., Ford, K., Cassin, A.,Natera, S., and Bacic, A. (2007).Increased abundance of proteinsinvolved in phytosiderophore pro-duction in boron-tolerant barley.Plant Physiol. 144, 1612–1631.

Perfus-Barbeoch, L., Leonhardt, N.,Vavasseur, A., and Forestier, C.(2002). Heavy metal toxicity: cad-mium permeates through calciumchannels and disturbs the plant waterstatus. Plant J. 32, 539–548.

Rakwal, R., Agrawal, G. K., andYonekura, M. (1999). Separa-tion of proteins from stressedrice (Oryza sativa L.) leaf tis-sues by two-dimensional polyacry-lamide gel electrophoresis: inductionof pathogenesis-related and cellularprotectant proteins by jasmonic acid,UV irradiation and copper chloride.Electrophoresis 20, 3472–3478.

Requejo, R., and Tena, M. (2006).Maize response to acute arsenic tox-icity as revealed by proteome anal-ysis of plant shoots. Proteomics 6,S156–S162.

Ritter, A., Ubertini, M., Romac, S.,Gaillard, F., Delage, L., Mann, A.,

et al. (2010). Copper stress pro-teomics highlights local adaptation oftwo strains of the model brown algaEctocarpus siliculosus. Proteomics 10,2074–2088.

Rodríguez-Celma, J., Rellan-Alvarez, R.,Abadia, A., Abadia, J., and Lopez-Millan, A. F. (2010). Changes inducedby two levels of cadmium toxicity inthe 2-DE protein profile of tomatoroots. J. Proteomics 73, 1694–1706.

Salt, D. E., and Rauser, W. E. (1995).MgATP-dependent transport of phy-tochelatins across the tonoplast of oatroots. Plant Physiol. 107, 1293–1301.

Sanita Di Toppi, L., and Gabbrielli,R. (1999). Response to cadmium inhigher plants. Environ. Exp. Bot. 41,105–130.

Saravanan, R. S., and Rose, J. K. (2004).A critical evaluation of sample extrac-tion techniques for enhanced pro-teomic analysis of recalcitrant planttissues. Proteomics 4, 2522–2532.

Sarowar, S., Kim, Y. J., Kim, E. N., Kim,K. D., Hwang, B. K., Islam, R., et al.(2005). Overexpression of a pepperbasic pathogenesis-related protein 1gene in tobacco plants enhances resis-tance to heavy metal and pathogenstresses. Plant Cell Rep. 24, 216–224.

Sarry, J. E., Kuhn, L., Ducruix, C.,Lafaye, A., Junot, C., Hugouvieux, V.,et al. (2006). The early responses ofArabidopsis thaliana cells to cadmiumexposure explored by protein andmetabolite profiling analyses. Pro-teomics 6, 2180–2198.

Schneider, T., Schellenberg, M., Meyer,S., Keller, F., Gehrig, P., Riedel,K., et al. (2009). Quantitative detec-tion of changes in the leaf-mesophylltonoplast proteome in dependencyof a cadmium exposure of barley(Hordeum vulgare L.) plants. Pro-teomics 9, 2668–2677.

Semane, B., Dupae, J., Cuypers, A.,Noben, J. P., Tuomainen, M., Ter-vahauta, A., et al. (2010). Leafproteome responses of Arabidopsisthaliana exposed to mild cadmiumstress. J. Plant Physiol. 167, 247–254.

Sharma, S. S., and Dietz, K. J. (2009).The relationship between metal tox-icity and cellular redox imbalance.Trends Plant Sci. 14, 43–50.

Sharmin, S. A., Alam, I., Kim, K. H.,Kim, Y. G., Kim, P. J., Bahk, J. D., et al.(2012). Chromium-induced physio-logical and proteomic alterations inroots of Miscanthus sinensis. Plant Sci.187, 113–126.

Tuomainen, M. H., Nunan, N., Lehes-ranta, S. J., Tervahauta, A. I.,

Hassinen, V. H., Schat, H., et al.(2006). Multivariate analysis of pro-tein profiles of metal hyperaccumu-lator Thlaspi caerulescens accessions.Proteomics 6, 3696–3706.

van Hoof, N. A., Hassinen, V. H.,Hakvoort, H. W., Ballintijn, K. F.,Schat, H., Verkleij, J. A., et al. (2001).Enhanced copper tolerance in Silenevulgaris (Moench) Garcke popula-tions from copper mines is associatedwith increased transcript levels of a2b-type metallothionein gene. PlantPhysiol. 126, 1519–1526.

Van Loon, L., and Van Strien, E. (1999).The families of pathogenesis relatedproteins, their activities, and compar-ative analysis of PR-1 type proteins.Physiol. Mol. Plant Pathol. 55, 85–97.

Van Loon, L. C. (1997). Induced resis-tance in plants and the role ofpathogenesis-related proteins. Eur. J.Plant Pathol. 103, 753–765.

Vannini, C., Marsoni, M., Domingo, G.,Antognoni, F., Biondi, S., and Bra-cale, M. (2009). Proteomic analysisof chromate-induced modificationsin Pseudokirchneriella subcapitata.Chemosphere 76, 1372–1379.

Verbruggen, N., Hermans, C., and Schat,H. (2009). Mechanisms to cope witharsenic or cadmium excess in plants.Curr. Opin. Plant Biol. 12, 364–372.

Villiers, F., Ducruix, C., Hugouvieux,V., Jarno, N., Ezan, E., Garin,J., et al. (2011). Investigating theplant response to cadmium expo-sure by proteomic and metabolomicapproaches. Proteomics 11, 1650–1663.

Wang, L., Zhao, J.-Y., Wu, S.-M.,Pan, J.-L., Huang, Z.-B., Wu, Z.-K.,et al. (2011). No statistic corre-lation between superoxide dismu-tase and peroxidase activities andaluminum-induced lipid peroxida-tion in maize, implying limitedroles of both enzymes in preven-tion against aluminum-induced lipidperoxidation. Am. J. Plant Sci. 2,156–164.

Wang, W., Vinocur, B., Shoseyov,O., and Altman, A. (2004). Roleof plant heat-shock proteins andmolecular chaperones in the abioticstress response. Trends Plant Sci. 9,244–252.

Welch, R. M., and Norvell, W. A.(1999) “Mechanisms of cadmiumuptake, translocation and depositionin plants,” in Cadmium in Soils andPlants, eds M. J. McLaughlin andB. R. Singh (Dordrecht: The KluwerAcademic Publishers), 125–150.

Yadav, S. K., Singla-Pareek, S. L., Ray,M., Reddy, M. K., and Sopory, S.K. (2005). Methylglyoxal levels inplants under salinity stress are depen-dent on glyoxalase I and glutathione.Biochem. Biophys. Res. Commun. 337,61–67.

Yang, Z., and Chu, C. (2011). “Towardsunderstanding plant response toheavy metal stress,” in Abiotic Stress inPlants – Mechanisms and Adaptations,eds A. Shanker and B. Venkateswarlu(Shanghai: InTech). Available at:http://www.intechopen.com/books/abiotic-stress-in-plants-mechanisms-and-adaptations

Yang, Q., Wang, Y., Zhang, J., Shi, W.,Qian, C., and Peng, X. (2007). Iden-tification of aluminum-responsiveproteins in rice roots by a proteomicapproach: cysteine synthase as a keyplayer in Al response. Proteomics 7,737–749.

Zhang, H., Lian, C., and Shen, Z.(2009). Proteomic identification ofsmall, copper-responsive proteins ingerminating embryos of Oryza sativa.Ann. Bot. 103, 923–930.

Zhen, Y., Qi, J. L., Wang, S.S., Su, J., Xu, G. H., Zhang,M. S., et al. (2007). Comparativeproteome analysis of differentiallyexpressed proteins induced by Al tox-icity in soybean. Physiol. Plant 131,542–554.

Conflict of Interest Statement: Theauthors declare that the research wasconducted in the absence of any com-mercial or financial relationships thatcould be construed as a potential con-flict of interest.

Received: 29 November 2012; paperpending published: 13 December 2012;accepted: 24 December 2012; publishedonline: 25 January 2013.Citation: Hossain Z and Komatsu S(2013) Contribution of proteomic studiestowards understanding plant heavy metalstress response. Front. Plant Sci. 3:310.doi: 10.3389/fpls.2012.00310This article was submitted to Frontiers inPlant Proteomics, a specialty of Frontiersin Plant Science.Copyright © 2013 Hossain and Komatsu.This is an open-access article distributedunder the terms of the Creative CommonsAttribution License, which permits use,distribution and reproduction in otherforums, provided the original authors andsource are credited and subject to anycopyright notices concerning any third-party graphics etc.


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