Soil metals, chloroplasts, and secure crop production: a review

675

26 Heavy Metals and Plastid Metabolism

Katalin Solymosi and Martine Bertrand

26.1 INTRODUCTION

Heavy metals (HMs) are in general defined as metals with a specific gravity greater than 5.0 or with high atomic mass. Often, toxicity is associated with their definition; however, several HMs are necessary for the proper functioning and metabolism of living organisms. Such elements essential for plant development are Cu, Co, Fe, Mn, Mo, Ni, and Zn. One type of plant stresses is nutrient deficiency, with which most plants can barely cope; therefore their growth and crop productivity are impaired when the phytoavailability of any of these metals is low. Unfortunately, in several agricultural fields, essential-metal deficiency is a serious concern, especially in the case of Fe and Zn (Guerinot 2000). Another plant stress is represented by the excess of essential HMs and by the presence of nonessential metals. These nonessential HMs (e.g., Cd, Cr, Hg, Pb) are in general toxic or neutral for plant metabolism. The increased presence of HMs in air, soil, and water is also a global problem that represents a growing threat to the environment and to humankind and requires immediate attention. There are hundreds of natural and anthropologic sources of HMs, including industry, atmospheric deposition, use of agrochemicals, and waste disposal. The pollutants can enter plants via various mechanisms and in this way can easily reach intracellular compartments of plants, such as plastids. In this chapter, the stress caused by essential-HM deficiency and by excess

CONTENTS

26.1 Introduction .......................................................................................................................... 67526.2 Metals Inside Plants .............................................................................................................. 676

26.2.1 Metal Transporters .................................................................................................... 67626.2.2 Interactions between Different Metals .....................................................................680

26.3 Metals and Plastid Metabolism............................................................................................. 68126.3.1 Impact of Unbalanced Metals on Nongreen Plastids ............................................... 68226.3.2 Impact of Unbalanced Metals on Photosynthesis ..................................................... 685

26.3.2.1 Ultrastructural Alterations in Chloroplasts ...............................................68826.3.2.2 Molecular and Metabolic Alterations in Chloroplasts

under Heavy-Metal Deficiency .................................................................. 69126.3.2.3 Molecular and Metabolic Alterations in Chloroplasts

under Heavy-Metal Excess ........................................................................ 69326.3.3 Some Unusual Phenomena Associated with Heavy-Metal Stress ............................ 698

26.3.3.1 When the Excess of a Metal Alleviates the Stress Caused by Another Metal ....................................................................................... 698

26.3.3.2 When Nonessential Metals Added at Low Concentrations Have a Stimulating Effect ..........................................................................699

26.4 Conclusion ............................................................................................................................700Abbreviations ................................................................................................................................. 701References ...................................................................................................................................... 701

676 Handbook of Plant and Crop Stress

of HMs on plastid metabolism is discussed. In addition, the interesting phenomena of HM stress alleviation by the excess of another HM and the stimulatory effect of low concentration stressors on photosynthesis is briefly reviewed.

26.2 METALS INSIDE PLANTS

For healthy plant growth and development, a range of HMs must be acquired from the soil, trans-ported along the plant, distributed, and compartmentalized in different tissues and cells. Clearly, membrane transport systems are likely to play a key role in these events. The genes encoding these plant nutrient transporters appear to be transcriptionally regulated by a feedback mechanism that reduces their expression when the plant reaches an optimal level of nutrition (reviewed by Amtmann and Blatt 2009, Burkhead et al. 2009, Kawachi et al. 2009). In most terrestrial plants, metals are absorbed by the roots and transported via the xylem into the aerial parts. Essential HMs are in gen-eral highly mobile and therefore approximately one third of them is translocated to the aboveground organs, while two-thirds are retained in the roots (e.g., Kawachi et al. 2009). However, when present or added in excess, most of the surplus essential metal(s) also accumulates in the roots, similar to most nonessential or toxic metals (e.g., Brunner et al. 2008, Zehra et al. 2009), but rarely the con-taminating metal principally accumulates in the leaves (e.g., Vázquez et al. 1990). This way, in most cases, the root plastids are the first targets of HM toxicity or deficiency. Therefore, root plastids are in general less prone to metal deficiency, but more often affected by high levels of metals.

Aquatic plants and some epiphytes absorb the essential nutrients via their whole surface from the water or the air, respectively. In these cases, HMs may reach the plastids directly. Similarly, atmospheric deposition of pollutants contributes to toxic metal levels in aerial plant parts (leaves, fruits, flower parts, and stems) of terrestrial plants, and in this case, chloroplasts or other types of plastids (i.e., chromoplasts, amyloplasts) can be directly affected by relatively high concentrations of HMs, although not much is known about the uptake mechanisms and transportation of airborne metal pollutants within plant tissues.

Nonessential metals (Cd, Ag, and Pb), generally intrude into plant cells or into organelles at the expense of essential inorganic ions on account of similar properties, such as ionic radii (Perfus-Barbeoch et al. 2002), for example, Cd uses Ca ion channels to enter plant cells.

26.2.1 METAL TRANSPORTERS

After the first phase of extracellular metal adsorption—rapid and nonspecific binding of the cations to the negatively charged cell wall components and mobilization of the soil-bound HMs by secre-tion of organic acids by the roots—metals have to enter the symplasm by metal transporters or ion channels (Table 26.1). Most of the metals enter cells as cationic elements (e.g., Zn2+), whereas others cross the plasma membrane as anionic groups (e.g., AsO4

3−) or included in small organic compounds (e.g., methyl-mercury, CH3Hg+) (reviewed by Kucera et al. 2008). The HMs are taken up as hydrated ions or in metal–chelate complexes through channel proteins and/or carrier proteins. These include the (1) heavy-metal (P1b-type or CPx-type) ATPases (HMA) that can pump a variety of essential and nonessential HMs across the plasma membrane; (2) the natural resistance associated macrophage proteins (NRAMP), which are H+-coupled transporters implicated in the transport of divalent ions (e.g., Cu2+, Fe2+, Mn2+, and Cd2+); (3) the cation diffusion facilitators (CDFs) involved in Zn2+, Co2+, Mn2+, and Cd2+ transport (Williams et al. 2000); (4) the ZRT, IRT-like protein (ZIP) family transporting mostly Fe2+, Mn2+, Cd2+, and Zn2+ (Cohen et al. 1998, Guerinot 2000, reviewed by Grotz and Guerinot 2006); (5) the cation/H+ antiporters exchanging Na+ or H+ to Ca2+ or Cd2+; and (6) ATP-binding cassette (ABC) transporters that have a role in intracellular transport of Cd, Fe, or Mn (Table 26.1, reviewed by Clemens 2001, Hall and Williams 2003, Kucera et al. 2008, Poirier et al. 2008). Although these transporters are rather specific for a single element, their specificity is not absolute, and other metals can also be transported (Table 26.1). However, knowledge about

Heavy Metals and Plastid Metabolism 677

TAB

LE 2

6.1

Hea

vy-M

etal

Tra

nspo

rter

s an

d Io

n C

hann

els

Cha

ract

eriz

ed I

n V

ivo

or I

n V

itro

and

Loc

aliz

ed E

xper

imen

tally

or

Onl

y C

ompu

ter

Pred

icte

d to

Dif

fere

nt P

lant

Mem

bran

es

Car

rier

Typ

eC

arri

er

Prop

osed

Met

al S

peci

fici

ty

(++

Ind

icat

es t

he P

rim

arily

Tra

nspo

rted

Met

al)

Loca

lizat

ion

Ref

eren

ces

Cd

Co

Cu

FeM

nN

iPb

Zn

AB

C-t

ype

tran

spor

ter

STA

1+

MB

riat

et a

l. (2

007)

Cat

ion

diff

usio

n fa

cilit

ator

(C

DF)

ZA

T1/

MT

P1 a

nd M

TP3

+T

Van

der

Zaa

l et a

l. (1

999)

, Bro

adle

y et

al.

(200

7), P

uig

and

Pena

rrub

ia (

2009

),

and

Kaw

achi

et a

l. (2

009)

ShM

TP1

+T

Del

haiz

e et

al.

(200

3)

Cat

ion/

H+ a

ntip

orte

rsC

AX

2 (C

a)+

+T

Hir

schi

et a

l. (2

000)

CA

X4

(Ca)

++

TC

heng

et a

l. (2

002)

Con

serv

ed c

oppe

r tr

ansp

orte

r (C

OPT

)C

OPT

1, C

OPT

2+

PMSa

ncen

on e

t al.

(200

3), P

uig

et a

l. (2

007)

, an

d B

urkh

ead

et a

l. (2

009)

CO

PT?

+T

Yru

ela

(200

9)

CO

PT?

++

+PE

Sanc

enon

et a

l. (2

003)

, Bur

khea

d et

al.

(200

9), a

nd Y

ruel

a (2

009)

Cyc

lic n

ucle

otid

e g a

ted

chan

nels

(sh

aker

type

)C

NG

C1

NtC

BP4

? ++

+ +PM PM

Sunk

ar e

t al.

(200

0)A

razi

et a

l. (1

999)

and

Tal

ke e

t al.

(200

3)

Hea

vy-m

etal

AT

Pase

s (H

MA

)H

MA

1+

++

++

+PE

Seig

neur

in-B

erny

et a

l. (2

006)

, Yru

ela

(200

9), a

nd K

im e

t al.

(200

9)

HM

A2

++

++

++

+PM

Ere

n an

d A

rgüe

llo (

2004

)

HM

A3

++

++

+T

Pilo

n et

al.

(200

9) a

nd M

orel

et a

l. (2

009)

HM

A4

++

+PM

Ere

n an

d A

rgüe

llo (

2004

)

HM

A5

+PM

, GA

ndre

s-C

olas

et a

l. (2

006)

and

Yru

ela

(200

9)

(con

tinu

ed)


TAB

LE 2

6.1

(con

tinu

ed)

Hea

vy-M

etal

Tra

nspo

rter

s an

d Io

n C

hann

els

Cha

ract

eriz

ed I

n V

ivo

or I

n V

itro

and

Loc

aliz

ed E

xper

imen

tally

or

Onl

y C

ompu

ter

Pred

icte

d to

Dif

fere

nt P

lant

Mem

bran

es

Car

rier

Typ

eC

arri

er

Prop

osed

Met

al S

peci

fici

ty

(++

Ind

icat

es t

he P

rim

arily

Tra

nspo

rted

Met

al)

Loca

lizat

ion

Ref

eren

ces

Cd

Co

Cu

FeM

nN

iPb

Zn

HM

A6/

PAA

1+

++

?PE

Shik

anai

et a

l. (2

003)

, Abd

el-G

hany

et a

l. (2

005)

, Pui

g et

al.

(200

7), a

nd Y

ruel

a (2

009)

HM

A8/

PAA

2+

PISh

ikan

ai e

t al.

(200

3), P

uig

et a

l. (2

007)

, an

d B

urkh

ead

et a

l. (2

009)

OsH

MA

9+

++

PML

ee e

t al.

(200

7)

RA

N1/

HM

A7

+E

R, P

GY

ruel

a (2

009)

and

Kim

et a

l. (2

009)

Low

-affi

nity

cat

ion

tran

spor

ter

TaL

CT

1 (C

a)+

PMSc

hach

tman

et a

l. (1

997)

, Ant

osie

wic

z an

d H

enni

g (2

004)

, Woj

as e

t al.

(200

7),

and

Szcz

erba

et a

l. (2

009)

Mag

nesi

um-s

elec

tive

ion

chan

nel

MG

T1

(Mg)

++

++

+PM

Li e

t al.

(200

1)

Mg2+

(Z

n2+)/

H+ a

ntip

orte

rM

HX

(M

g)+

++

TSh

aul e

t al.

(199

9) a

nd B

erez

in e

t al.

(200

8)

NR

AM

PN

RA

MP1

++

++

PEC

urie

et a

l. (2

000)

, Tho

min

e et

al.

(200

0),

Gro

tz a

nd G

ueri

not (

2006

), a

nd P

uig

et a

l. (2

007)

NR

AM

P3 T

cNR

AM

P3+

++

+T

Gro

tz a

nd G

ueri

not (

2006

) an

d O

omen

et a

l. (2

009)

Heavy Metals and Plastid Metabolism 679N

RA

MP4

TcN

RA

MP4

++

++

TH

all a

nd W

illia

ms

(200

3), G

rotz

and

G

ueri

not,

(200

6), a

nd O

omen

et a

l. (2

009)

Olig

opep

tide

tran

spor

ter

(OPT

)Y

SL1,

YSL

3C

u-N

AFe

-NA

Zn-

NA

PMW

ater

s an

d G

rusa

k (2

008)

Zm

YS1

Fe-P

SPM

Cur

ie e

t al.

(200

1)

Perm

ease

PIC

1+

PED

uy e

t al.

(200

7) a

nd P

uig

and

Pena

rrub

ia

(200

9)

Vac

uola

r ir

on tr

ansp

orte

rV

IT1

+T

Kim

et a

l. (2

006)

and

Bri

at e

t al.

(200

7)

ZR

T, I

RT-

like

prot

eins

(Z

IP)

IRT

1+

++

++

++

PMC

ohen

et a

l. (1

998)

, Con

nolly

et a

l. (2

002)

, Fo

dor

(200

6), P

uig

et a

l. (2

007)

, and

Mill

s et

al.

(200

8)

IRT

3+

+PM

Bro

adle

y et

al.

(200

7)

ZIP

2 an

d Z

IP4

+±

PM?

Bur

khea

d et

al.

(200

9)

ZIP

4+

+PE

Gro

tz e

t al.

(199

8), H

all a

nd W

illia

ms

(200

3),

and

Gro

tz a

nd G

ueri

not (

2008

)

Not

es:

Car

rier

s ar

e lis

ted

in a

lpha

betic

ord

er. F

or m

ore

deta

ils s

ee th

e ci

ted

refe

renc

es. +

+ in

dica

tes

the

mai

n tr

ansp

orte

d H

M, a

nd w

hen

the

tran

spor

ter

is p

rim

arily

res

pons

ible

for

the

tran

s-po

rt o

f ano

ther

(non

-hea

vy) m

etal

, the

sym

bol o

f the

met

al is

indi

cate

d in

par

enth

esis

aft

er th

e na

me

of th

e ca

rrie

r. A

bbre

viat

ions

: AB

C, A

TP-

bind

ing

cass

ette

; ER

, end

opla

smic

retic

u-lu

m;

G,

Gol

gi;

M,

mito

chon

dria

l m

embr

ane;

NA

, ni

cotia

nam

ine;

PE

, pl

astid

env

elop

e; P

G,

post

-Gol

gi m

embr

anes

; PI

, pl

astid

inn

er m

embr

anes

; PM

, pl

asm

a m

embr

ane;

PS,

ph

ytos

ider

opho

re; T

, ton

opla

st.

? Q

uest

ion

mar

ks in

dica

te u

nces

tain

ties.


these transporters is still rather scarce and many of them are only characterized in vitro. Some of them are expressed in an organ-specific manner, and their intracellular localization also varies, i.e., some are located in the plasma membrane, others are involved in other endomembranes such as chloroplast or mitochondrial envelopes, Golgi, and tonoplast membranes (Table 26.1, reviewed by Hall and Williams 2003). As a consequence, some of them have a role in metal uptake, extracellular detoxification, or long-distance transportation of HMs within the plants (from those located in the plasma membrane); others have roles in the sequestration of excess HMs in the vacuole or in their remobilization from this organelle (these are found in the tonoplast) or in metal delivery to other intracellular compartments.

While transporters require active transport through symporters and antiporters, and can transport ions against an electrochemical potential gradient, passive ion transport occurs through channels, which are membrane proteins with ion-selective pores that allow ion movement down an electro-chemical gradient. Among cationic channels, some are not highly selective and can therefore par-ticipate in the transport of different toxic cations (reviewed by Demidchik et al. 2002, Table 26.1).

26.2.2 INTERACTIONS BETWEEN DIFFERENT METALS

The different HMs can interact in the soil. Positive and negative synergisms, competition, protec-tion, and sequential additivity are observed among the interactions. The nature of interactions varies considerably with concentration levels, soil pH, soil texture, level of soluble Ca in soil, presence of salinity, differential distribution in soil of the metals present in high quantities, presence or absence of chelating agents, soil organic matter levels, and other factors (reviewed by Wallace et al. 1992). HMs bind organic ligands with different stability constants or may form precipitates with inorganic anions in the soil solution of the rhizosphere. This way, they might mutually influence each other’s solubility or compete for different binding ligands, including those secreted out by the plant to improve the solubility of essential metals (e.g., phytosiderophores for Fe; Delhaize and Ryan 1995, Hinsinger et al. 2003). As illustrated in Table 26.1, some HMs use the same ion channels, metal transporters or chelators, and therefore they have an impact on each other’s uptake and intracellular concentration. Therefore, toxic HMs often cause reduced productivity and biomass in crop plants indirectly, by inducing essential-metal deficiency in the plants.

It is generally assumed that for nonessential elements such as Cd, there are no specific uptake mechanisms. Cd ions compete with nutrients such as K, Ca, Mg, Fe, Mn, Cu, Zn, and Ni (Table 26.1, reviewed by Pál et al. 2006, Clemens et al. 2009). Some data suggest that Cd can be taken up via the phytosiderophore pathway as well (reviewed by Reichman and Parker 2005, Fodor 2006). This outlines that, besides alleviating nutrient deficiency of Fe, Zn or Ni, phytosiderophores also increase the bioavailability of toxic metals and thus increase the potential for food-chain transfer hazards for them (e.g., for Cd), and they also increase the competition between Fe and other metals, leading then to physiological Fe-deficiency. In Cd-treated leaves, Cd can enter guard cells via Ca channels (Perfus-Barbeoch et al. 2002). It was also observed that Pb can enter the cells via Ca and Ni transport systems (Table 26.1, Arazi et al. 1999, Sunkar et al. 2000, Wojas et al. 2007). However, data are relatively scarce in the literature about the uptake of nonessential HMs.

Besides the competition of metals in the soil and during metal uptake in the roots, HMs also com-pete in their translocation from the roots to the shoots. Since very little metal in plants is assumed to exist as free ions, several small organic molecules have to be implicated in metal ion homeostasis as metal ion ligands or chelators, in order to improve acquisition and transport of metal ions with low solubility and to enhance immobilization for metal tolerance and storage. Citrate, mugineic acid, avenic acid, deoxymugineic acid (Suzuki et al. 2008) and nicotianamine have been shown to participate in the intra- and intercellular transport of essential metals such as Cu, Fe, Mn, Ni, or Zn (reviewed by Fodor 2006, Puig et al. 2007, Chen et al. 2009, Yruela 2009). In vitro, nicotianamine is able to form stable complexes with Mn, Fe, Co, Zn, Ni, and Cu, in increasing order of affinity (Curie et al. 2009). The pH stability of these complexes suggests their occurrence in symplasm or


apoplasm, indicating that nicotianamine should complex Cu, Fe, and Zn in the phloem, and Cu and Zn in the xylem for their translocation from roots to shoots (reviewed by Yruela 2009).

The Zn/Cd-transporting ATPases, HMA2 and HMA4, essential for root-to-shoot Zn transloca-tion, facilitate the transport of Cd (Table 26.1, Wong and Cobbett 2009). Citrate was found to be the principal compound chelating metals in the xylem sap (Cd: reviewed by Fodor 2006, Hasan et al. 2009; Pb: reviewed by Fodor 2002, 2006).

Finally, after their xylem unloading, essential HMs must enter the symplasm of the cells in the aerial parts of the plant by different membrane transport systems (Table 26.1) and have to reach the intracellular compartment where they will be used. The mechanisms of intracellular HM trafficking is still not very well understood. In the next part of the chapter, we review the effect of unbalanced metal concentrations on the structure and function of different plastids.

26.3 METALS AND PLASTID METABOLISM

The plastid is a unique, semiautonomic organelle characteristic of photosynthetic eukaryotic cells and evolved from the endosymbiosis of free-living cyanobacteria with an ancient eukaryotic cell (reviewed by Solymosi and Schoefs 2008). All plants contain plastids. These organelles are wide-spread within the plants, because with a few exceptions, all cells possess plastids in one form or another. Despite their diversity, plastids have several common features. Their boundary to the cyto-plasm is a double membrane called the plastid envelope, which encircles the protein-rich stroma. The outer membrane is permeable to molecules up to a molecular mass of ca. 6 kDa due to the pres-ence of porins, while the inner membrane is highly selective and contains different membrane trans-porters (e.g., Table 26.1, reviewed by Weber et al. 2005, Johnson et al. 2006, Aronsson and Jarvis 2008). Besides the more or less developed inner membrane system, the plastids often contain spheri-cal bodies that contain lipids, carotenoids, plastoquinone, and proteins and others that are called plastoglobuli that contain lipids, carotenoids, plastoquinone, and proteins (Austin et al. 2006). In addition, the plastids contain nucleoids (DNA-containing structures), procaryotic ribosomes and, as semiautonomic organelles, they can synthesize at least part of their proteins. Different inclusions are also frequently seen in the stroma of the plastids. One example for them is the Fe-containing phytoferritin or simply ferritin (reviewed by Briat et al. 1999).

Depending on their physiological function, chemical composition and internal structure, the plastids are divided into different groups (reviewed by Solymosi and Schoefs 2008). Chloroplasts are present in many different types of cells and organs (i.e., in ripening or mature fruits, in green colored parts of flowers like the calyx and the gynoecium, in green stems and leaves). Their pres-ence is essential because they provide energy and oxygen to the biosphere via photosynthesis. Their inner membrane system is laterally segregated into two major functional domains, the appressed (stacked) granal membranes and the interconnected, non-appressed stromal membranes (reviewed by Solymosi and Schoefs 2008).

Besides chloroplasts, several nongreen plastid types exist and are developmentally interrelated. Proplastids are characteristic in meristematic cells (e.g., in the root and shoot apical meristems), and in dedifferentiated and/or reproductive cells (reviewed by Solymosi and Schoefs 2008). Proplastids are small and have only a poorly developed inner membrane structure. They can differentiate into any other plastid type. Under natural light conditions, and in photosynthesizing organs, they dif-ferentiate into chloroplasts. However, in angiosperm plants, in the absence of light, normal chloro-plast development is impaired, and proplastids differentiate to so-called etioplasts (e.g., Solymosi et al. 2004, 2006a, 2007, reviewed by Solymosi and Schoefs 2008). The formation of etioplasts is a natural phenomenon observed in different crops (e.g., cabbage heads—Solymosi et al. 2004) and in seeds germinating in the soil in agricultural systems (e.g., sunflower: Solymosi et al. 2007; bean: Schoefs and Franck 2008, reviewed by Solymosi and Schoefs 2008). These plastids contain special inner membrane system consisting of lamellar prothylakoids and a paracrystalline membrane net-work called prolamellar body.


During senescence of the tissues, and also before defoliation of the leaves, the conversion of chloroplasts to the so-called gerontoplasts (or senescing chloroplasts) can be observed (reviewed by Thomas 1997). During this transformation, chlorophyll (Chl) is degraded, the inner membrane system of the chloroplasts is disorganized and large, often electron-transparent plastoglobuli appear (e.g., Solymosi et al. 2004).

The leucoplasts are colorless plastids with poorly developed inner membranes; they are special-ized in storage of either starch (amyloplasts) or lipids (elaioplasts) or proteins (proteinoplasts) and function therefore as storage organelles. These plastids are heterotrophic and convert photosyn-thates derived from source tissues into storage compounds. Amyloplasts are characteristic of the parenchymatic tissues of storage organs (tubers, rhizomes, roots, fruits, seeds) but can also be found in root cap cells, where they are associated with geotropism (reviewed Solymosi and Schoefs 2008).

The chromoplasts accumulate carotenoids and are responsible for the bright yellow, orange, and red colors of petals, fruits like tomatoes, pepper, roseberry and for that of some roots, i.e., carrot (reviewed by Solymosi and Schoefs 2008).

Thus, in addition to photosynthesis, plastids harbor many more vital biosynthetic functions, such as nitrogen and sulfur assimilation or the biosynthesis of fatty acids, (aromatic) amino acids, lip-ids, pigments (Chls and carotenoids), purines, pyrimidines and secondary metabolites including terpenoids, and other important compounds used in pharmaceutical or perfume industry. In con-sequence, these functions require an active solute exchange across the outer and inner envelope membranes surrounding the chloroplast stroma (reviewed by Weber et al. 2005). Metal transport proteins in both membrane systems thus provide a bottleneck to the control of metal homeostasis in the chloroplast as well as in the plant cell (Table 26.1).

The transition metals Fe, Cu, and Mn play a vital role in photosynthetic electron transport in chloroplasts and in stroma-located reactions of CO2 fixation (Table 26.2). Plastid localized Fe and Cu/Zn superoxide dismutases scavenge reactive oxygen species (ROS). In addition, Zn is known to function as a cofactor (in RNA polymerase, Zn finger domains) in plastid transcription (Table 26.2), while among others, Fe is required for heme and for Fe-S clusters (Cornah et al. 2002) and for enzymes of Chl biosynthesis (Myśliwa-Kurdziel and Strzałka 2002, Duy et al. 2007). Several other enzymes functioning in other plastid types also require HMs (Table 26.2).

Among all plastid types, the effect of metal stress and also the functioning of metal uptake machineries are best characterized in chloroplasts, but there are also a few data indicating changes induced by excess metals in other plastid types (for details see below). Both essential-metal defi-ciency and excess influence plastid metabolism. Similarly, various concentrations of nonessential metals also affect plastid structure and function. In this chapter, these processes are briefly sum-marized. Since the Chl concentration may fundamentally influence the functioning of the photo-synthetic apparatus and thus affect the whole plant metabolism, it is a really important factor in assessing the impact of metal stress in chloroplasts and on plant productivity. One of the most usual symptoms of metal deficiency or metal excess is chlorosis, i.e., the decrease of the Chl content. This further outlines, that plastids—and especially chloroplasts—are central targets in metal stress.

26.3.1 IMPACT OF UNBALANCED METALS ON NONGREEN PLASTIDS

As discussed earlier, except in the case of airborne HMs in terrestrial or epiphytic plants, or water pol-luting elements for aquatic plants, HMs generally first affect the cells of roots or other underground organs (such as potato tubers, onion bulbs) and may interact with their plastids. Unfortunately, there are almost no data about the effect of HM deficiency or excess on nongreen plastids.

Although excess HMs and essential-metal deficiency also interfere with the metabolism of non-green plastids, much less data are available on structural and metabolic alterations of these plastids (Table 26.3) than chloroplasts. For instance, besides chloroplasts, heme biosynthesis mostly occurs in root plastids and etioplasts, and is affected by Fe-deficiency (Cornah et al. 2002). Nongreen plastids play important roles in several important plant metabolic processes including amino acid


biosynthesis, hormone synthesis, sugar homeostasis, storage of different metabolites, carotenoid synthesis, and secretion. The secondary metabolite production of nongreen plastids is important in medicinal plants and crops also for human health and nutrition. Often, edible parts of crops contain nongreen plastids (e.g., carrot roots contain chromoplasts; celery, potato tubers, onion bulbs, gar-lic, and radish contain amyloplasts and/or proplastids; the inner tissues of cabbage heads, avocado and different cucumber fruits contain etioplasts or etio-chloroplasts) (reviewed by Solymosi and Schoefs 2008). However, molecular interactions of these plastids and the effect of metals on plastid metabolism in these organs remain poorly understood. Root plastids seem to be a primary target

TABLE 26.2Nonexhaustive List of Key Molecules Requiring Essential Metals in the Chloroplast

Metals Proteins References

Cu Cu/Zn-superoxide dismutase Grace (1990)

Cytochrome oxidase Hänsch and Mendel (2009)

Plastocyanin Abdel-Ghany (2009)

Polyphenol oxidase Kieselbach et al. (1998)

Fe Ascorbate peroxidase Raven et al. (1999)

Cytochrome b6-f Raven et al. (1999)

Cytochrome c6 Raven et al. (1999)

Ferredoxin Tognetti et al. (2007)

Ferredoxin–thioredoxin reductase Duy et al. (2007)

Ferritin Briat et al. (1999)

Ferrochelatase Cornah et al. (2002)

Fe-SOD Allen (1995)

Glutamine-2-oxo-glutarate amido transferase Duy et al. (2007)

NADPH-plastoquinone oxidoreductase Raven et al. (1999)

Nitrite reductase Briat and Vert (2004)

Pheophorbide a oxygenase Duy et al. (2007)

Sirohydrochlorin ferrochelatase Duy et al. (2007)

Sulfite reductase Duy et al. (2007)

Tic55 Duy et al. (2007)

Mg Chlorophylls Shaul (2002)

Glutathione synthetase Shaul (2002)

Mn Isocitrate dehydrogenase Elias and Givan (1977)

Malic enzyme Takeuchi et al. (2000)

Mn-SOD Grace (1990)

Phenylalanin ammonia lyase Nishizawa et al. (1979)

Water oxydase Grace (1990)

Mo Aldehyde oxidase Weigel et al. (1986)

Sulfite oxidase Eilers et al. (2001)

Xanthine dehydrogenase Borner et al. (1986)

Zn Carbonic anhydrase Randall and Bouma (1973)

Cu/Zn-SOD Grace (1990)

Enzymes involved in RNA editing Hänsch and Mendel (2009)

Metalloendopeptidase Moberg et al. (2003)

Stromal processing peptidase Hänsch and Mendel (2009)

Zn finger Sasaki et al. (1989)

Zn-metalloprotease FtsH Bailey et al. (2001)

Zn-protease degrading RUBISCO Bushnell et al. (1993)


of metal excess. The detailed review of Barceló and Poschenrieder (2006) came to the conclusion that except for Cd, no visible ultrastructural damage is observed in the organelles of the roots, but the metals rather disturb the polar zonation of the organelles within the cells. Changes in the amyloplasts and their arrangement in root columella cells (Table 26.3) may directly influence root gravitropism and growth direction and seem to be associated with, for example, Al stress–induced root growth defects. Sometimes, alterations in plastid shape were also reported, for example, in Cr6+ and 99Tc-treated roots, where amoeboid plastids occurred in the roots (Table 26.3, Vázquez et al. 1987, 1990). Similarly, metal stress altered sugar metabolism and often, changes in the starch contents of plastids have been reported (Table 26.3). The Cd-induced ferritin accumulation in bean root plastids may be related to disturbed Fe homeostasis of these plants (Vázquez et al. 1992). Cu excess–induced the formation of dark globular inclusions of unknown nature in plastids (Panou-Filotheou and Bosabalidis 2004).

Another important, but poorly studied field is the effect of metals on plastid differentiation, on the biosynthesis of the photosynthetic apparatus and on chloroplast biogenesis. These studies are even more important, as seedlings germinating in polluted soil have to cope with metal stress at this level, and seedlings that fail to develop functionally active chloroplasts do not survive. Chloroplast differentiation may proceed directly from proplastids, or in case of agricultural systems, seedlings germinating from seeds buried in the soil often develop etioplasts before reaching the soil surface (reviewed by Solymosi and Schoefs 2008). Therefore, studies related to the impact of excess met-als on proplastids, etioplasts or young chloroplasts differentiating from these two plastid types are crucial to understand the molecular interactions of metals with these organelles and also to enhance seedling survival in polluted areas.

TABLE 26.3Examples of Alterations in the Ultrastructure of Nongreen Plastids Caused by Excess of Heavy Metals

Metal Species Plastid Alterations References

Cd Bean (Phaseolus vulgaris) Decreased starch content and reduced internal membrane system in root plastids

Barceló et al. (1988)

Ferritin-like deposits in root plastids Vázquez et al. (1992)

Maize (Zea mays) No ultrastructural effect on etioplasts and on etioplast–chloroplast conversion

Ghoshroy and Nadakavukaren (1990)

Soybean (Glycine max) Disruption of the prolamellar body structure in etioplasts, retarded etioplast–chloroplast conversion (delayed grana formation, swollen intrathylakoidal spaces in greening plastids)

Ghoshroy and Nadakavukaren (1990)

Cr (VI) Bean (Phaseolus vulgaris) Amoeboid plastids in root tip cells, appearance of amyloplasts in upper parts of the roots

Vázquez et al. (1987)

Increased starch content in etio-chloroplasts of stems

Cu Oregano (Origanum vulgare)

Amyloplast-leucoplast transition is induced in roots, decrease in starch content, dark globular inclusions and many small droplets of an electron translucent substance

Panou-Filotheou and Bosabalidis (2004)

Hg Wheat (Triticum aestivum)

Changes in the unit cell size of prolamellar bodies (PLBs) of etioplasts, formation of vesicles on the PLB surface

Solymosi et al. (2006b)

99Tc Common bean (Phaseolus vulgaris)

Amoeboid plastids, different forms of plastid inclusions (“engulfment” of cytoplasm or a mitochondrion)

Vázquez et al. (1990)


Several metals (Cd, Na, K and Hg) influence the etioplast to chloroplast transformation (Table 26.3). These elements induce slight structural alterations in etioplast structure, which are in some respect similar to those observed in chloroplasts (see later, i.e., swelling of intrathylakoidal space, formation of vesicles, regularly spotted bodies indicating possible osmotic stress). Chl biosyn-thesis and etioplast–chloroplast transition (the reorganization of the inner membrane system and the development of the thylakoids and the photosynthetic apparatus) is impaired under different metal stresses under in vitro conditions with short-term, high-concentration treatments (excess Cd: Ghoshroy and Nadakavukaren 1990; excess Na and K: Abdelkader et al. 2007; excess Hg: Solymosi et al. 2006b). This might indirectly indicate that these processes may be also partially inhibited in the light.

26.3.2 IMPACT OF UNBALANCED METALS ON PHOTOSYNTHESIS

Chloroplasts are key organelles for plant development, growth and biomass production because of their ability to produce sugars via photosynthesis. On the other hand, photosynthesis is a process that requires several metalloproteins containing different HMs (Table 26.2). The dif-ferent essential metals present in plastids, besides being structural constituents of various mol-ecules, are also important in modulating reactions or cellular processes, or in maintaining the ion homeostasis of the organelles or the cells. For instance, plastids seem to have a role in Ca (Seigneurin-Berny 2000), Cu (Abdel-Ghany 2009), and Fe (Izaguirre-Mayoral and Sinclair 2005, 2009) storage, Fe being often stored in the metabolically inactive form of phytoferritin, which prevents photooxidation reactions caused by “free” metal ions (Briat et al. 1999, Arosio and Levi 2002, Ravet et al. 2009).

As discussed earlier, plants possess different uptake systems to transport essential metals into the plastids (Table 26.1). Part of these transporters can also deliver nonessential elements. However, the knowledge about plastid metal transporters in respect to metal stress is relatively scarce and it is difficult to determine the exact concentration of metals inside chloroplasts. Most of such studies have been done on algae, in which the accumulation of different metals can be easily observed in thylakoid membranes (reviewed by Barceló and Poschenrieder 2006), but they are not relevant for terrestrial plants. Only rough estimations exist, that assume that 1% of total Cd content of plants is probably transported to plastids (reviewed by Siedlecka and Krupa 1999). Some data about metal concentrations in chloroplasts are summarized in Table 26.4. Unfortunately, even if available, often other literature data about plastid metal contents are not comparable, because they are expressed in different and non-interconvertible units or on different basis (e.g., Cd: Ramos et al. 2002; Cu: Baszyński et al. 1978; Zn, Mg, Cu: Kim et al. 2009) or were determined only in thylakoids (e.g., Mn: Lidon and Teixeira 2000a, Lidon et al. 2004) or were measured after different durations of metal treatments (e.g., Ramos et al. 2002).

TABLE 26.4Intraplastidial Concentration of Some Essential Metals

Metal Concentration within the Plastid References

Ca Total: 4–23 mM, free: few μM Johnson et al. (2006)

Cu 60 μM Joyard and Douce (1976)

Fe 0.13 mM Joyard and Douce (1976)

K 150 mM Neuhaus and Wagner (2000)

Mg 5 mM Neuhaus and Wagner (2000)

Mn 33 μM Joyard and Douce (1976)

Zn 0.13 mM Joyard and Douce (1976)


At the same time, the mode of HM pollution is also important in determining the interactions of the metals with plastids. In the leaves of lettuce seedlings treated with Cd solutions for 16 days through their roots, the lowest intracellular Cd concentration (6–16 μg g−1 FW, 12%–14% of total Cd content of leaves) was found in the chloroplasts (Ramos et al. 2002). Interestingly, the Cd content of the chloroplasts was 10–22 μg g−1 FW (8%–9% of total leaf Cd content) when lettuce leaves were incubated for 24 h in Cd-containing solutions, a model experiment mimicking airborne metal pol-lution (Ramos et al. 2002). In both cases, most Cd was accumulated in the apoplasm. These studies indicate that, in case of airborne metals, atmospheric metal deposition may have a stronger influ-ence on plastid metabolism than soil pollution (Ramos et al. 2002).

Developing methods of electron microscopy combined with analytical techniques such as energy dispersive x-ray microanalysis (EDXA), laser microprobe mass analysis (LAMMA), electron energy loss spectroscopy (EELS), Synchrotron x-ray fluorescence (SXRF) microbeam analyses, secondary ion mass spectrometry or cytochemical methods are powerful tools to characterize primary mecha-nisms of metal toxicity and tolerance on the cellular and molecular level (reviewed by Barceló and Poschenrieder 2006). Similarly, atomic force microscopy, laser scanning optical microscopy using confocal microscopy or multiphoton excitation provide important information about metal stress on the cellular and tissue level. New methods are also under development (e.g., laser-induced breakdown spectroscopy and laser-ablation inductively coupled plasma mass spectrometry—Kaiser et al. 2009). Unfortunately, data about microlocalization of metals in plastids with these imaging methods are rela-tively scarce, probably because of the low concentrations accumulating in these organelles being under the actual detection limit of these methods. However, direct interaction of the metals with chloroplast membranes and metabolism cannot be excluded even in cases when the amounts of metals were below the detection limit in the plastids. Therefore, we overview some data that reported direct connections between the absence or the presence of metals in plastids, and observed metabolic alterations in vivo.

Chl biosynthesis requires several metals; therefore, decreased Chl content is a general symptom of essential-HM deficiency (Cu: Burkhead et al. 2009, Hänsch and Mendel 2009, Yruela 2009; Fe: Puig et al. 2007; Mn: Simpson and Robinson 1984, González and Lynch 1999, Yu et al. 1999, Henriques 2003, 2004; Zn: Singh et al. 2005). There are almost no data about metal concentrations in plastids under HM deficiency. Different estimations indicate that about 80% of foliar Fe is pres-ent in the plastids (Terry and Low 1982, Thoiron et al. 1997), and in Arabidopsis green tissues, 40% of Fe is found in the thylakoids (reviewed by Briat et al. 2007), so these organelles can be expected to be one of the first targets of Fe-deficiency. Chlorotic leaves of Fe-deficient plants have yellowish color because of decreased Chl content due to impaired Chl biosynthesis and the symp-toms appear first in the interveinal areas (Thoiron et al. 1997, Misra et al. 2006, Mahmoudi et al. 2007, Timperio et al. 2007). Some authors have postulated that Fe-deficiency induced chlorosis is related to decreased Fe and Chl concentrations in thylakoid lamellae, and this way to retarded or disturbed thylakoid formation rather than to inhibited Chl biosynthesis per se (Terry and Low 1982). Moreover Fe-deficiency would impact photosystem I (PSI) functioning because of the reduced num-ber of Fe-S clusters (Doan et al. 2003, Duy et al. 2007).

The effect of excess HMs is often also reflected by reduced plant growth and chlorosis. In these cases, often the observed phenotypic alterations could be directly linked with the presence of the excess metals in the chloroplasts. For instance, the toxic effects of Ni on chloroplast structure of cabbage plants (Molas 1997, 2002) are in good agreement with the observed Ni accumulation inside the organelles. The histochemical techniques of Ni localization show that in cabbage plants the important sites for Ni accumulation in the leaf are mesophyll cells located on leaf edges, near the vascular bundles and intervascular bundles affected with chlorosis (Molas 2002). The chloroplast, the cell walls and the nucleus are the most important Ni-accumulation sites at the cellular level.

In rice plants treated with increasing concentrations of Mn, the accumulation of this metal was found in the leaves and thylakoid membranes, which indicates that this element is highly mobile, enters the chloroplasts and accumulates up to a certain level in these organelles (Lidon et al. 2004). Similarly, it has been demonstrated that large part of Mn quantity entering the cytoplasm moves and


binds on the outer side of thylakoid membranes of chloroplasts (González and Lynch 1999, Lidon and Teixeira 2000b, Lidon et al. 2004) affecting their structure and photosynthesis. Moreover leaf chlorosis induced by excess Mn was positively correlated to Mn content of chloroplasts (González and Lynch 1999). Therefore, in this case, the observed ultrastructural symptoms might be at least partially linked to direct interactions of Mn with thylakoid membranes.

Chloroplasts are one of the main sites of Cu accumulation (Maksymiec 1997). Data indicate that under Cu treatment, this ion can enter the cytoplasm and the plastids also by Ca channels espe-cially in younger plants and at the initial stages of the stress (Maksymiec and Baszyński 1999). Cu can bind directly to the thylakoids where it may induce oxidative stress (Figure 26.1, reviewed by

GSSG

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redox metals

Oxidized

redox metals

Lipoxygenase

PUFA

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Metals

Cd, Co, Cu

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Cd, Cu, Hg

Ni, Pb, Zn(k)

As, Cd, Cr

Cu, Hg, Ni, Zn

PAA2/

HMA8

PC

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2

FIGURE 26.1 Oxidative stress and defense reactions occurring in the chloroplast, when excess of metal is present. (a) Depletion of sulfhydryl groups by metals on reduced glutathione (GSH) regenerated by glutathione reductase (GR) from its oxidized form (GSSG); (b) Activation of lipoxygenase by metals; (c) Peroxidation of polyunsaturated fatty acids (PUFA) to saturated fatty acids (RCOOH + R′COOH) in membranous phospho-lipids; (d) Cu transporter PAA2/HMA8; (e) Electron transfer from the reaction center (RC) to the acceptor (Acc) of PSI or PSII; (f) univalent oxygen reduction by PSII; (g) Electron transfer through ferredoxin (Fd) and NADP+ (specific to PSI); (h) Fenton and Haber–Weiss reactions: one-electron oxidoreductions performed by redox metals leading to hydroxyl radicals (OH•); (i) Spontaneous and/or superoxide dismutase (SOD) cata-lyzed disproportionation of superoxides (O2

−). Superoxide radicals generated by PSI and PSII are dismutated by the membrane-bound m-SOD, and the formed hydrogen peroxide (H2O2) is reduced to water by the thy-lakoid-bound ascorbate peroxidase (AP). Monodehydroascorbate (MDHA) is reduced back to ascorbate (A) by the photoreduced ferredoxin (Fd). O2

− ions escaped from the thylakoid system are scavenged by a stromal s-SOD; ( j) Xanthophyll cycle associated to the enzymes epoxidase and de-epoxidase, preventing superox-ide formation at high light intensity; (k) Inactivation of plastocyanin (PC) by nonessential metals after its release from the membrane or the release of its Cu; (l) Inactivation of Calvin cycle enzymes; (m) Chelation of metals by phytochelatins either imported from the cytoplasm or synthesized through the activity of a putative phytochelatine synthetase (PCS). → activation by metals; ┤ inhibition by metals.


Maksymiec 1997). Under excess Cu, the Cu content of leaf chloroplasts showed more than tenfold increase on the protein or Chl content basis (Baszyński et al. 1988), which indicates that this ele-ment might directly interact with chloroplast components or might induce oxidative stress directly.

In case of poorly mobile, nonessential metals such as Cr, present in the soil, the ultrastructural changes induced in the plastids and chlorosis are probably due to indirect effects of the metal only, because they appear without substantial increase in the metal concentration in the leaves (e.g., Moustakas et al. 1997, reviewed by Barceló and Poschenrieder 2006).

As Table 26.2 lets it assume, each essential HM has specific functions and is needed in appropri-ate amounts within chloroplasts. Thanks to efficient ion homeostasis, plant development and func-tioning may be optimal also under moderate HM stress, but large metal unbalance is a disaster for plastids both on the structural and metabolic levels.

26.3.2.1 Ultrastructural Alterations in ChloroplastsAs some HMs are crucial for the functioning of several proteins and structural components of chloro-plasts (Table 26.2), it is not surprising that too low concentrations of HMs may cause chlorosis, functional alterations of photosynthesis and other ultrastructure. Before discussing the effects of toxic concentra-tions of metals on chloroplast structure, we briefly review the symptoms of essential-metal deficiency.

Ultrastructural alterations are easy to be observed, but are more complicated to be interpreted on the metabolic level. Since unbalanced metal concentrations can be the result of several phe-nomena (including competition of essential and nonessential metals for transport systems in plants and this way to indirect effects), and to interaction of different metals with each other on several levels, including metabolic levels, the most reliable data about the direct ultrastructural effects of essential-metal deficiency are those that were obtained in nutrient transporter mutants impaired in chloroplast metal uptake or homeostasis (Henriques et al. 2002, Song et al. 2004, Duy et al. 2007). The disturbances in thylakoid biosynthesis can be in this case directly related to nutrient deficiency, outlining the importance of essential metals in plastid differentiation and in the maintenance of the active photosynthetic apparatus.

Some recently characterized knockout mutants impaired in Fe and Zn uptake (irt1) or in their transport into the plastids (pic1) show several ultrastructural alterations at the chloroplast level. Irt1 mutants have reduced thylakoid system and stacking (maximum 2–3 thylakoids per grana), smaller starch grains and increased number of plastoglobuli (Henriques et al. 2002). Pic1 knockouts have unchanged Fe level, but changed ion homeostasis, Cu content, and their internal membrane system is significantly reduced (Duy et al. 2007). No grana, just simple and parallely arranged thylakoids or even no thylakoid characterize the plastids. Vesicle formation and increased ferritin content are also characteristic in these mutants (Duy et al. 2007).

There are several other data in the literature that describe the ultrastructural alterations of chloroplasts of plants grown under essential-HM deficient conditions (Figure 26.2, Table 26.5).

1 2 3 4 5 6 7

(b) (c)(a)

8

FIGURE 26.2 Scheme summarizing the most important ultrastructural alterations of chloroplasts (b) induced by essential metal deficiency (a) or by excess of essential or nonessential metals (c). Note the swelling of the organelle and the alterations of the different structural elements of chloroplasts (1: plastoglobuli, 2: swollen intrathylakoidal space, 3: peripheral vesicles, 4: starch grains, 5: grana, 6: regularly spotted bodies, 7: dark deposits on the thylakoid surface 8: ferritin clusters).

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Chloroplasts are often swollen in such plants (Zn deficiency: Chen et al. 2008), or sometimes have smaller size than in plants grown under optimal metal concentrations (Fe deficiency: Platt-Aloia et al. 1983; Mn deficiency: Papadakis et al. 2007). Rarely, the disruption of the plastid envelope has been observed in such plants (Zn-deficient rice: Chen et al. 2008). Abnormal chloroplasts with almost no grana and disrupted or disorganized internal membranes (Cu deficiency: Thoiron et al. 1997, Briat et al. 1999, Henriques 2003; Mn deficiency: Possingham et al. 1964, Weiland et al. 1975, Izaguirre-Mayoral and Sinclair 2005; Zn deficiency: Thomson and Weier 1962, Henriques 2001, Chen et al. 2008), the swelling (dilatation) of the intrathylakoidal space (Cu deficiency: Baszyński et al. 1988, Henriques 1989; Fe deficiency: Thoiron et al. 1997; Mn deficiency: Weiland et al. 1975, Izaguirre-Mayoral and Sinclair 2005, Papadakis et al. 2007; Zn deficiency: Chen et al. 2008), the appearance of clusters of unusual, peripheral vesicles (Fe deficiency: Platt-Aloia et al. 1983, Briat et al. 1999; Mn deficiency: Mercer et al. 1962, Possingham et al. 1964), increased number (and size) of plastoglobuli (Cu deficiency: Henriques 1989; Fe deficiency: Ji et al. 1984, Henriques 2003; Mn deficiency: Weiland et al. 1975, Izaguirre-Mayoral and Sinclair 2005; Zn deficiency: Chen et al. 2008) are usual symptoms of metal deficiency. The starch contents of plastids also vary with differ-ent essential-HM availability; however, these changes are not consistent, i.e., Fe deficiency causes decreased starch content in apple chloroplasts (Ji et al. 1984), while starch content increases in chlo-roplasts of Fe-deficient pecan (Henriques 2003). Decreased starch size and content is observed in plastids of Mn-deficient lemon (Papadakis et al. 2007) and soybean (Weiland et al. 1975, Izaguirre-Mayoral and Sinclair 2005).

Several data are available about the ultrastructural alterations caused by the excess of various HMs added in various treatments, under various experimental conditions and in various plant spe-cies. However, interestingly the symptoms are quite similar (Table 26.5). Most studies have been conducted under laboratory conditions (e.g., hydroponic cultures, etc.), but sometimes the plastid ultrastructure of plants grown in naturally contaminated soils has been also described (wheat plants grown in Cu-contaminated soils: Eleftheriou and Karataglis 1989). These plants contain a reduced number and a reduced size of chloroplasts, less developed plastid inner membrane system; they also have impaired grana formation, decreased starch content and decreased number of plastoglobuli (Eleftheriou and Karataglis 1989).

In some cases, different steps of plastid ultrastructural alterations were distinguished as a func-tion of increasing concentrations and/or duration of metal exposure (e.g., Na: Rahman et al. 2000; Zn: Doncheva et al. 2001). In this latter case, the first symptoms of Zn stress observed at lowest HM concentrations is the disintegration of stromal thylakoids, and the reduction of grana number, swol-len thylakoids (i.e., the swelling of the intrathylakoidal space), decreased starch content, increase in size and number of plastoglobuli appear subsequently with increasing Zn concentrations (Doncheva et al. 2001).

In the leaves of plants grown in experimental conditions with excess of HMs, the number of chloroplasts may decrease (Table 26.5), which is probably due to metal interference with chloroplast replication (reviewed by Kucera et al. 2008). Besides this phenomenon, damage to chloroplasts is the most frequently observed ultrastructural effect of toxic metals in leaves. The inhibition of normal plastid development in HM-treated plants may be indicated by the appearance of amoeboid plastids observed occasionally (Vázquez et al. 1990) and is further supported by the fact that, in gen-eral, chloroplasts of young leaves are more affected by metal stress than old leaves (e.g., Maksymiec et al. 1995, Skórzyńska-Polit and Baszyński 1997, Maksymiec and Baszyński 1999). However, con-tradictory data have been also published (Barceló et al. 1988), and these observations might not be generalized, they also depend on the developmental stage at which metal pollution occurs. The most common ultrastructural symptoms are the swelling of the organelle, distortion of thylakoids leading to the loss of the parallel arrangement of the thylakoid membranes, reduction or increase of the thylakoid surface area, and swelling of the intrathylakoidal space (Table 26.5, reviewed by Barceló and Poschenrieder 2006). There are contradictory data about the changes in the starch content upon HM excess, which may increase or decrease in the plastids (Table 26.5). Often, the


senescence of the chloroplasts, i.e., the chloroplast-gerontoplast conversion, is induced by the excess of metals (Table 26.5), which is not only indicated by the degeneration of grana and disorders in the thylakoid system but also by the increase in the number and size of plastoglobuli. These symptoms may be a result of metal-induced alteration of the hormone balance (e.g., Cd: Vassilev et al. 2004, Rodríguez-Serrano et al. 2009, reviewed by Maksymiec 1997, for more details about all metals, see Fodor 2002, Maksymiec 2007) leading to enhanced senescence. Most studies have dealt with plastid ultrastructure in leaves, however, some data indicate that chloroplasts in the green stems are also similarly damaged by excess of metals (Barceló et al. 1988).

The careful comparison of the ultrastructural effects induced by essential-HM deficiency and excess of HMs (Figure 26.2, Table 26.5) indicates that besides several similar structural alterations, there are specific differences. Regularly spotted bodies, appearance of dark electron dense deposits at grana surfaces and ferritin were only related to the presence of excess HMs and might therefore be specific for this type of stress, while unusual peripheral vesicles appear under metal-deficient conditions (Figure 26.2, Table 26.5). Disruption of the plastid envelope was more often observed in plastids of plants treated with excess HMs.

These results further outline the complicated interrelations and the difficulties in interpreting the effects of HMs at the molecular and cellular level. The observed effects have been mostly interpreted as the excess or deficiency of HMs on membranes or osmotic disturbances. However, it is unclear to what extent these ultrastructural effects are due to direct toxicity of the metal ions in the chloroplast, to metal-induced membrane disturbances, to metal-induced enhancement of ROS, to osmotic problems, or to consequences of metal-induced deficiency of essential nutrients. In the following sections we briefly review the possible reasons of the observed ultrastructural alterations.

26.3.2.2 Molecular and Metabolic Alterations in Chloroplasts under Heavy-Metal Deficiency

Essential HMs are needed for normal plastid ultrastructure, homeostasis, and functioning (i.e., they represent functional components of the thylakoids and the stroma, take part in plastid protein synthesis, DNA replication, … etc., Table 26.1), therefore, it is not surprising that their structure is strongly influ-enced in plants grown under nutrient-deficient conditions (Figure 26.2, Table 26.5). Nutrient-deficiency symptoms are usually expressed as reduced growth, biomass, and physiological functions. Chl biosyn-thesis requires several metals (Table 26.2); therefore decreased Chl content is a general symptom of essential-HM deficiency (Cu: Burkhead et al. 2009, Hänsch and Mendel 2009, Yruela 2009; Fe: Puig et al. 2007; Mn: Simpson and Robinson 1984, González and Lynch 1999, Yu et al. 1999, Henriques 2003, 2004; Zn: Singh et al. 2005). Not only because of the decreased Chl content, but due to direct interactions with photosynthetic reactions, the rate of CO2 fixation and biomass also decrease (e.g., Zn deficiency: Srivastava et al. 1997; Cu-deficiency: Droppa et al. 1984, Maksymiec 1997).

Oxidative stress is also one of the most important components of mineral-nutrient-deficiency stresses (e.g., Zn, Mn, Fe, Cu, B, Mg, and K) (Yu et al. 1999). This is not surprising as some micronutrients, such as Zn, Fe, and Cu are important components of ROS scavenging systems of plastids (Cu/Zn and Fe superoxide dismutases, Table 26.2), therefore, plants deficient in these met-als may exhibit symptoms of oxidative stress. Below, we briefly overview some metabolic processes impacted by the deficiency of the most important essential HMs.

Fe-deficiency affects not only Chl biosynthesis (Duy et al. 2007, reviewed by Myśliwa-Kurdziel and Strzałka 2002), but the functioning of the photosynthetic electron chain, the Calvin cycle (Siedlecka and Krupa 1996, reviewed by Myśliwa-Kurdziel and Strzałka 2002), plastid protein import (Duy et al. 2007), enzymes of chloroplast-localized nitrogen fixation machinery (Briat and Vert 2004), sulfur assimilation, siroheme biosynthesis, amino acid metabolism (Duy et al. 2007), and Fe-involving antioxidative response (Allen 1995) (Table 26.2). The different molecular altera-tions of the photosynthetic apparatus induced by Fe-deficiency (Sárvári 2005, Briat et al. 2007) and the adaptation mechanisms of photosynthesis under Fe starvation (Sharma 2007) are summarized

https://www.researchgate.net/publication/6468389_PIC1_an_Ancient_Permease_in_Arabidopsis_Chloroplasts_Mediates_Iron_Transport?el=1_x_8&enrichId=rgreq-a0f1edf3-4638-49ab-bfd2-b3258befcd42&enrichSource=Y292ZXJQYWdlOzIzNjI3MjM5MztBUzoxNTkwOTgyOTMyMDI5NDRAMTQxNDk0MzM5NzM4Ng==


elsewhere. Some recently characterized mutants impaired in Fe uptake (irt1) or in its transport into the plastids (pic1) have similar ultrastructural damage as reported in Fe-deficient plants (Figure 26.2) (Henriques et al. 2002, Duy et al. 2007). The disturbances in thylakoid biosynthesis and plas-tid differentiation can be in this case directly related to nutrient deficiency, outlining the importance of essential metals in plastid differentiation and in the maintenance of the active photosynthetic apparatus.

Zn deficiency also leads to chlorosis and decreased photosynthetic activity in leaves (Cakmak 2000, Henriques 2001, Wang and Jin 2005, Chen et al. 2008). Leaves become then light sensitive. The leaves of Zn-deficient sugar beet plants have disorganized chloroplasts with senescence process leading to cell death and, ultimately, to necrotic blade lesions, thus reducing the photosynthetically-active leaf area, which explains the lower CO2 fixing capacity and decreased biomass of Zn-deficient plants (Henriques 2001). The observed symptoms are probably associated with oxidative stress (for a review see Cakmak 2000) and decreased antioxidant enzyme levels (e.g., Cu/Zn-superoxide dis-mutase = Cu/Zn-SOD) in Zn-deficient leaves (Wang and Jin 2005, Chen et al. 2008, reviewed by Cakmak 2000, Rengel 2006). In Zn-deficient chickpea plants, disturbances in stomatal conductance and water status were also observed (Khan et al. 2004). The reduced growth and productivity of Zn-deficient plants are often associated with decreased levels of indole-3-acetic acid (reviewed by Cakmak 2000). Zn deficiency also alters membrane lipid composition and fatty acid saturation and leads to lipid peroxidation. Zn deficiency induces a decrease in the activity of carbonic anhydrase, which catalyzes the reversible reaction of CO2 hydration and is therefore accompanied by reduced photosynthetic rates (reviewed by Rengel 2006).

Cu is a crucial plastid component involved in photosynthetic electron transport (plastocyanin) but is also indispensable for photosystem II (PSII) and light-harvesting complex II (LHC II) and for the functioning of the antioxidant enzyme, Cu/Zn-SOD (reviewed by Maksymiec 1997). Fifty percent of total plastidic Cu is found in plastocyanin (reviewed by Hänsch and Mendel 2009). Therefore, it is not surprising that photosynthetic functions are highly impaired under Cu-deficiency. Decreased plastocyanin, Chl, and carotenoid contents observed in Cu-deficient leaves can be responsible for the lower rates of photosynthesis (Baszyński et al. 1978, Henriques 1989, Shikanai et al. 2003, reviewed by Burkhead et al. 2009). PSI activity and cyclic photophosphorylation (Baszyński et al. 1978) as well as PSII activity are also targets of Cu-deficiency (reviewed by Myśliwa-Kurdziel and Strzałka 2002). Cu deficiency is often accompanied with oxidative stress probably due to decreased Cu/Zn-SOD levels (reviewed by Rengel 2006). The lipid composition of Cu-deficient chloroplasts is altered (reviewed by Barón et al. 1995). The galactolipid content decreases and the fatty acid unsaturation levels are also altered in the different lipid fractions (they increase in galacto-lipids). A secondary effect of Cu deficiency can be insufficient water transport caused by a decrease in cell wall formation and lignification in several tissues, including xylem tissue (for a review, see Burkhead et al. 2009).

Mn-deficiency symptoms include reduced growth, pale young leaves that subsequently develop interveinal chlorosis, and ultimately small necrotic spots (Yu et al. 1999, Henriques 2003, Henriques 2004). Mn deficiency also induces oxidative stress that could be partially prevented in transgenic tobacco plants overexpressing Mn-SOD in chloroplasts (Yu et al. 1999). Mn-deficient plants show the loss of most, but not all, functional PSII reaction centers in grana, with no altera-tion in light-harvesting complex of PSI, which is linked to the disruption of the oxygen-evolving complex (Simpson and Robinson 1984). However, upon Mn resupply, the leaves become able again to control Mn levels after 2 days. Mn deficiency depresses leaf photosynthetic capacity primarily by reducing the number of PSII units in spinach leaves (e.g., Simpson and Robinson 1984). Recent investigations in pecan leaves have shown that the reduced number of PSII units per leaf area unit is achieved by decreasing the number of chloroplasts, but not the number of PSII per individual chloroplast, and that the remaining PSII possess photochemical abilities similar to those of control leaves (Henriques 2003, 2004), their Mn content being similar to that of chloroplasts isolated from control plants (Henriques 2004).


26.3.2.3 Molecular and Metabolic Alterations in Chloroplasts under Heavy-Metal ExcessSeveral visible symptoms have been observed in plants grown in the presence of excess essential or nonessential HMs. Leaf expansion is inhibited, leaf tissues can become deformed and chloro-sis often occurs (e.g., Cd: Djebali et al. 2005, Ebbs and Uchil 2008, Ben Ghnaya et al. 2009; Cr: Vázquez et al. 1987; Cu excess: Baszyński et al. 1988, Ciscato et al. 1997, Panou-Filotheou et al. 2001; Mn excess: González and Lynch 1999, reviewed by Barceló and Poschenrieder 2006; Pb: Woźny et al. 1995; Zn excess: Doncheva et al. 2001, Ebbs and Uchil 2008, Wang et al. 2009). The inhibitory effect of excess metals on Chl biosynthesis (Myśliwa-Kurdziel and Strzałka 2002) and on photosynthesis (Fodor 2002, Myśliwa-Kurdziel et al. 2004, Sárvári 2005, Briat et al. 2007) is reviewed elsewhere in more detail. In this section, we briefly summarize the different direct and indirect effects of metals on plastid metabolism with emphasis on metal-specific effects and observations.

Clearly, the thylakoid membranes and plastids are much influenced by metal excess (Figure 26.2, Table 26.5); however, it is not clear if they are really the primary targets of metal stress—and there-fore, their alteration is responsible for the observed decrease in photosynthetic activity—or if they simply reflect the observed dramatic alterations of plastid metabolism. There are almost no data about the actual in organello concentration of excess metals, and therefore on the direct nature of their interactions. Furthermore several observed symptoms seem to be nonspecific. First, we briefly overview some examples for direct interactions of HMs with different plastid components (proteins, pigments, cofactors, and lipids), then we summarize data about metal-excess induced oxidative stress in plastids and the possible osmotic disturbances observed under excess metals. Finally, the essential metal (Fe, Cu, Zn, Mn)-deficiency induced by the excess of another HM is also discussed.

26.3.2.3.1 Molecules Impacted Directly by MetalsDirect interactions of metals with chloroplast metabolism could be often only studied in vitro at relatively high concentrations that are unlikely to occur in the organelles under natural conditions. These effects are summarized below.

26.3.2.3.1.1 Pigments and Other Small Plastid Metabolites Excess HMs may directly interact with pigments and may replace Mg in Chl in vivo or in vitro (Cd, Cu, Hg, Ni, Pb, Zn) (Figure 26.1) (Küpper et al. 1996, 1998, 2002, 2003). At high concentrations, different nonessential metals can also destabilize pigment precursors and may not substitute Mg, but induce its loss from the porphy-rin ring (e.g., protochlorophyllide: Solymosi et al. 2004). There is much less data about the direct interaction of excess HMs with carotenoids. In general, carotenoid levels seem to be affected indi-rectly by HM stress as these molecules have an important role in scavenging ROS induced by HMs (see below). In diatoms, the xanthophyll cycle was shown to be altered by Cd (Bertrand et al. 2001).

The observed decreased photosynthetic activity and disturbed plastid metabolism of HM-treated plants may be related to direct oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) by nonessential HMs (e.g., Cd: Figure 26.1, Böddi et al. 1995, reviewed by Pál et al. 2007; Hg: Lenti et al. 2002, Solymosi et al. 2004, Solymosi et al. 2006b), which might result in the inhibition of the activity of enzymes or metabolic processes that use NADPH as a hydrogen donor. Nonessential HMs have been also reported to cause high ATP content, and to change gene expression through DNA hypomethylation and DNA damage (reviewed by Poirier et al. 2008). This way, their carcino-genetic effect has also been reported (e.g., Monteiro et al. 2009). Hg-induced inhibition of photo-synthesis occurs probably by inducing a severe loss of adenylate pool and decreasing thus the rate of cyclic and non-cyclic photophosphorylation. Hg also decreases PSII associated reactions, O2 evolu-tion and CO2 fixation, probably due to the retardation of all ATP-dependent processes (reviewed by Romanowska 2002).

26.3.2.3.1.2 Lipids The observed changes in plastid and chloroplast membrane structure (Figures 26.1 and 26.2, Tables 26.3 and 26.5) might be due, at least partially, to membrane lipid


alterations in metal-exposed plants (reviewed by Devi and Prasad 2006). Nonessential HMs influ-ence the lipid composition, the saturation, or even the chain length of the fatty acids of membrane lipids. Several metals (e.g., Cd: Skórzyńska-Polit et al. 1998, Jemal et al. 2000, Nouairi et al. 2006; Pb: Stefanov et al. 1995) but also excess micronutrients (e.g., Cu: Maksymiec et al. 1992, Quartacci et al. 2000; Mn: Lidon et al. 2004) decrease the monogalactosyl-diacylglycerol (MGDG) content of thylakoid membranes. This change is explained by increased galactolipase activity (Skórzyńska and Baszyński 1993, Stefanov et al. 1995). Other lipid fractions have been also affected, but as MGDG and digalactosyl-diacylglycerol (DGDG) ratios influence membrane curvature, the changes in their ratios can explain the observed alterations in grana stacking and/or grana to stroma thyla-koid ratios under metal stress (Table 26.5, Figure 26.2). Similarly, the swollen intrathylakoidal space has been related to decreasing MGDG levels caused by increased galactolipase activity (Skórzyńska et al. 1991). MGDG is also required for proper functioning of photosynthesis (PSII complexes); therefore changes in this lipid fraction may have a detrimental effect on thylakoid functions and the total photosynthetic efficiency of plants. In addition, Ag, Cu, Pb, and Hg inhibit the plastidial phosphatidylcholine synthesis (Akermoun et al. 2002).

The amount of highly unsaturated fatty acids (especially 18:2 and 18:3) has been shown to increase after Cd (maize: Pál et al. 2007), Cu (spinach: Maksymiec et al. 1994), and Pb (spinach: Stefanov et al. 1995) treatment. However, contradictory data indicating lower degree of fatty acid unsaturation have been also reported in other plants treated with Cd (pepper: Jemal et al. 2000; tomato: Djebali et al. 2005; Brassica napus: Nouairi et al. 2006, Ben Ammar et al. 2007).

Increased fatty acid desaturation as well as decreased MGDG content can change membrane fluidity (Quartacci et al. 2000), which in turn leads to altered membrane physiological functions; it particularly influences the ionic permeability of the thylakoid membranes (for a review, Sandalio et al. 2001, see Devi and Prasad 2006). Different membrane permeabilities of the thylakoids might also explain the observed swelling of the intrathylakoidal space.

Membrane injuries of metal-treated plants (e.g., disruption of the envelopes, Table 26.5) are often related to increased peroxidation of membrane lipids caused by highly toxic free radicals (ROS) (reviewed by Devi and Prasad 2006, Maksymiec 2007). As, Cr, Cd, Cu, Hg, Ni, and Zn have all been shown to induce lipid peroxidation (Figure 26.1c) (e.g., Cd: Djebali et al. 2005, Skórzyńska-Polit and Krupa 2006; Hg: Cho and Park 2000) as well as several of the Fe and Cu compounds that can catalyze the Haber–Weiss and Fenton reactions (Babu et al. 2001). Cd directly affects the lipid structure around LHCII, leading to lipid peroxidation and the release of several pigment–protein complexes, oxygen-evolving complex (OEC) and plastocyanin (Figure 26.1k), then blocking further electron transport processes (for reviews, see Siedlecka and Krupa 1999, Pál et al. 2006). All these observations indicate that potentially toxic metals enter the plastids and can influence directly the different plastid components and then plastid metabolism.

26.3.2.3.1.3 Proteins One of the most often–reported direct toxic effects of metals on plastid proteins is attributed to the ability of some metals to bind to sulfhydryl-, histidyl-, and carboxyl-groups of proteins or enzymes, inducing therefore conformational changes resulting often in protein inactivation or disturbed function (Cd, Pb, Hg: Vallee and Ulmer 1972, Lenti et al. 2002, Solymosi et al. 2004; Cu: Maksymiec 1997, Yruela 2009).

Another possibility is that metals in excess may substitute essential metals in catalytic sites of enzymes (Figure 26.1). For instance, Hg can substitute Cu in plastocyanin (Radmer and Kok 1974), Co replaces Mg in the ribulose bisphosphate carboxylase oxygenase (RUBISCO), or Zn in transcrip-tion factors (reviewed by Poirier et al. 2008), and Cd replaces Ca in PSII reaction centre, causing the inhibition of PSII photoactivation (Faller et al. 2005, reviewied by Kucera et al. 2008). Cd can replace Zn and Ca in metalloenzymes (reviewed by Clemens et al. 2009). The substitution of Mn by Zn or Cd leads to the inactivation of OEC, and as a consequence, electron donation to PSII is inhibited (for reviews, see Bertrand and Poirier 2005, Pál et al. 2006, Kucera et al. 2008). Induced changes in the arrangement and structure of LHCII decrease the efficiency of excitation energy


capture by PSII and reduce the rate of photosynthetic oxygen evolution. Photophosphorylation rates decrease due to PSII dysfunctioning, without evidence for a direct inhibition of the ATP-synthase by the metal (reviewed by Kucera et al. 2008).

Hg ions can directly interact with some sites in the photosynthetic electron transport chain situated in D1 and D2 proteins, with the Mn cluster in the oxygen-evolving complex (reviewed by Romanowska 2002). Excess Cu inhibits several polypeptides of PSII and PSI (e.g., Lidon and Henriques 1993, Maksymiec et al. 1994). A primary site of Cu inhibition was identified on the antenna Chl a molecules of PSII (Lidon et al. 1993).

The enzymes RUBISCO, phosphoenolpyruvate carboxylase, alcohol dehydrogenase, glyceral-dehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and nitrate reductase have been described as sensitive to excess metal concentrations (Figure 26.2l) (reviewed by Siedlecka and Krupa 1999, Romanowska 2002, Myśliwa-Kurdziel et al. 2004, Pál et al. 2006, Kucera et al. 2008).

One possible explanation of the metal-induced chlorosis is that excess metals directly interact with enzymes of pigment biosynthesis. Other possibilities involve enhanced pigment degradation (partially due to oxidative stress), direct interaction with pigment precursors or cofactors required for the process, or metal-induced Fe- or Mg-deficiency as these essential metals have key roles in the biosynthetic processes (reviewed by Myśliwa-Kurdziel and Strzałka 2002). Preferential loss of pigments (e.g., Chl b in case of Zn and Cd stress—Ebbs and Uchil 2008) and decreased levels of carotenoids (e.g., excess Cu: Baszyński et al. 1988) are often reported under metal stress and can lead to impaired photosynthetic activities. Cr6+, Fe3+, and Hg2+ have been shown to directly inhibit one of the key enzymes of Chl biosynthesis, NADPH: protochlorophyllide oxidoreductase, in vitro (Lenti et al. 2002, Solymosi et al. 2004, Myśliwa-Kurdziel and Strzałka 2005). Cd also inhibits Chl biosynthesis directly through ALA dehydratase and protochlorophyllide reductase (in vitro: Böddi et al. 1995, Myśliwa-Kurdziel and Strzałka 2005, for reviews, see Myśliwa-Kurdziel and Strzałka 2002, Poirier and Bertrand 2005). However, other scientists have found that neither the synthesis nor the photoreduction of protochlorophyllide is influenced by Cd treatment in greening barley leaves (Horváth et al. 1996).

26.3.2.3.2 Metal-Induced Oxidative BurstHM ions block the electron flow in PSII, leading to the formation of excited triplet Chl (3Chl*), which can react with an oxygen molecule with triplet electronic configuration and by this way induces the formation of singlet oxygen. Oxidative stress leads to an imbalance in the regeneration and removal of ROS, including singlet oxygen (1O2), superoxide radical (O2−.), hydrogen peroxide (H2O2), and the most damaging and reactive hydroxyl radical (OH.), which can lead to further lipid peroxidation and can damage membranes, proteins, and nucleic acids, leading to altered membrane fluidity, loss of enzyme function, and genomic changes, respectively (reviewed by Kucera et al. 2008). Induction of free radicals and ROS by metals is very well documented and might be respon-sible for membrane injuries and some of the ultrastructural changes observed under metal stress (e.g., Babu et al. 2001, Zhang et al. 2005, Rodríguez-Serrano et al. 2009, reviewed by Sharma and Dietz 2008). However, some elements are considered to be redox-active metals (Cu, Fe) and can therefore directly elicit ROS generation (Gallego et al. 1996, Drazkiewicz et al. 2004), while others induce it only indirectly (Cd: Gallego et al. 1996, Pál et al. 2007; Cr and Cu: Yruela 2009; Hg: Cho and Park 2000; Mn: Lidon and Teixeira 2000a,b; Ni: Chen et al. 2009; Zn: Panda et al. 2003, Kawachi et al. 2009, reviewed by Sharma and Dietz 2008).

The different antioxidant enzyme systems (such as catalase localized into peroxisomes and plas-tids, SOD in the cytosol, mitochondria and plastids, peroxidases in vacuoles, cell walls and cytosol, and the ascorbate–gluthation cycle in several plant cell compartments) as well as carotenoids (and particularly the xanthophyll cycle) may protect the plants under oxidative stress and can there-fore be responsible for metal tolerance or can indicate metal stress (e.g., Drazkiewicz et al. 2004, reviewed by Kucera et al. 2008). Ascorbate takes part in growth processes, electron transport, pho-toprotection, regulation of photosynthesis, and preservation of the enzymatic activities that contain


prosthetic transition metal ions. Similarly, carotenoids that can quench the oxidizing ROS and the triplet state of Chl are often affected by metal stress (reviewed by Kucera et al. 2008). The carot-enoid content of metal-stressed leaves also changes, and particularly the xanthophyll cycle pigments that belong to a protection system for the photosynthetic apparatus from the nonphotochemical quenching of excited triplet Chl and from ROS (reviewed by Kucera et al. 2008). Therefore, high concentrations of antioxidative enzymes, together with enhanced pigment synthesis may allow a plant to completely overcome the harmful action of a toxic metal and to show normal healthy growth. However, the plant still accumulates fairly high amounts of the metal, a feature that can be useful for phytoremediation but dangerous in case of edible crops.

26.3.2.3.3 Metal-Induced Disturbances in Plastid Water and/or Ion HomeostasisIt is well documented that several HMs influence gas exchange and transpiration of plants (by influ-encing root hair formation, membrane permeability, number and diameter of vascular bundles, sto-matal conductance changes, and by inducing the closure of stomata) and therefore cause disturbances in respiration, in CO2 fixation, in the water and nutrient status of plants (e.g., Cd: Shi and Cai 2008, Nedjimi and Daoud 2009, Sayyad et al. 2009; Cr: Vázquez et al. 1987; Cu: Sayyad et al. 2009; Hg: Martínez-Ballesta et al. 2003; Mn: Lidon et al. 2004; Pb: Sayyad et al. 2009; Zn: Sayyad et al. 2009).

The inhibitory effect of metals on the dark phase of photosynthesis is also a complex phenom-enon. Increasing stomatal and mesophyll resistance leads to reduced CO2 uptake, because of a reduced number of stomata or stomatal closing (e.g., Moustakas et al. 1996, Shi and Cai 2008), which might directly inhibit crop production. Cr6+ treatment in bean has shown that this metal delayed or inhibited the differentiation of stomata on leaves (Vázquez et al. 1987). Similarly, under excess Ni, fewer stomata developed in cabbage leaves and many stomata were defective (Molas 1997). Decreased respiration and transpiration definitely alter the water and nutrient status of the plants. These data taken together, indicate that it is not surprising that sometimes the symptoms of metal excess—even at the plastid level—resemble those observed under osmotic disturbances or changes in water relations.

Some changes in the chloroplast shape and size (Figure 26.2, Table 26.5) or the disruption of the plastid envelope may indicate some osmotic disturbances of the membranes or changes in the envelope membrane permeability (Moustakas et al. 1997). In Cd- or Cu-stressed plants, chloroplasts often exhibited small, regularly spotted bodies, that were described as “pseudocrystalline bodies” (Cu: Ciscato et al. 1997) or “microtubule-like” structures (Cd: Ouzounidou et al. 1997) in wheat leaves. Similar structures have also been described in etioplasts of other species (barley: Wellburn et al. 1982, Wellburn 1984; wheat: Artus et al. 1990) and in greening plastids of Pb-treated bar-ley leaves (Woźny et al. 1995) or salt-stressed etiolated wheat leaves (Abdelkader et al. 2007) or wheat leaves grown in unusual environments (Solymosi, unpublished results). These spotted bod-ies might resemble the so-called prothylakoid bodies of unstressed plants (Wellburn 1984) or the so-called stromacenters formed in leaves dehydrated by plasmolysis, wilting or grown in windy areas (Gunning 1965, Gunning et al. 1968, Gunning and Steer 1975) and showing therefore some symptoms of water and osmotic imbalance.

It should be mentioned that direct osmotic effects are probable to occur in case of short-term treatments with very high, nonphysiological concentrations (0.1–1 M range). However, as most non-essential metal ions are toxic to plants from μM concentrations, the influence of such low concen-trations on the osmotic potential of the plants is probably negligible, but other, indirect effects on the water status of the plants are probable to occur (reviewed by Poschenrieder and Barceló 2006).

26.3.2.3.4 Deficiency of Essential Heavy Metals Induced by the Excess of Another Heavy Metal

It is well documented that indirect injury mechanisms caused by nonessential metals can be based on metal-induced deficiency of Fe, Mn, or other essential micro- or macronutrients. This process might be related to excess metal-induced changes in transpiration, root development, and as a


consequence to decreased water and nutrient uptake of plants, or to substitution or replacement of essential metals in metalloproteins (physiological essential-metal deficiency) or to competitive interactions with nutrient uptake and transport components.

According to the Irving–Williams series, the different metal ions can bind to organic ligands in a metal-binding site of a metalloprotein, metal chaperone or metal transporter with different affinities (Zn2+ < Cu+ > Cu2+ > Ni2+ > Co2+ > Fe2+ > Mn2+ > Mg2+ > Ca2+). At the same time, the binding affin-ity for a metal ion is also determined by other secondary factors such as the size of metal-binding-site cavity in a protein, the geometry of ligand atoms, and other characteristics, but normally each metal ion can be replaced by other metal ions downstream in the Irving–Williams series (reviewed by Yruela 2009). The potential for Zn, and especially Cu, to displace other metals is relatively high. This replacement is not only true for metal-containing proteins, important for plant functioning, for metal-containing molecules such as Chls (e.g., see Table 26.2), but also for all metal transporters and ion channels within the cells. Similarly, toxic metals may also substitute these ions. This way, it is quite evident that excess metals may induce nutrient deficiency. However, these interactions depend also on the treatment (i.e., if roots or leaves are treated), the form of the metal added, the plant mate-rial, and the experimental conditions (concentration of ions, pH, presence of chelators); therefore the results in the literature are difficult to compare (reviewed by Fodor 2002). Different chelators can have a strong influence on uptake, transportation, and/or on apoplasmic or non-apoplasmic accumu-lation of different interacting metals in the nutrient solution (e.g., Fodor et al. 2005).

One of the best known examples for nonessential metal-induced nutrient deficiency is the chloro-sis caused by Cd, which has been shown to be related to Fe-deficiency, rather than to the direct inhib-itory effect of Cd (e.g., Sárvári 2005, Fodor 2006, Pál et al. 2006). In plants grown on Fe-deficient nutrient solution, relatively more Cd was translocated into the shoot, and both PSs showed higher sensitivity to Cd (reviewed by Siedlecka and Krupa 1999). On the other hand, elevated Fe supply applied together or after Cd treatment could prevent most Cd effects (Siedlecka and Krupa 1996, Solti et al. 2008, reviewed by Siedlecka and Krupa 1999). Cd may also have indirect effects on PSI and influences the electron transport chain, ferredoxin-dependent NADP+ photoreduction, and Chl biosynthesis, by causing Fe-deficiency (reviewed by Siedlecka and Krupa 1999, Pál et al. 2006). Cd also lowers the Chl a/b ratio (Sárvári et al. 1999) due to stronger reduction of PSI than LHCII (Szegi et al. 2007). Cd reduced the amount of Chl-containing complexes in the order of PSI > LHCII > PSII core (Sárvári et al. 1999) similar to that observed in Fe-deficient plants. The indirect nature of the Cd-induced inhibition of the light phase of photosynthesis and of the different Chl–protein complexes is confirmed by the fact that in Cd-treated plants exhibiting all symptoms of Cd toxicity, the photosynthetic activity could be restored at least partially with the addition of Fe (Solti et al. 2008). Chl fluorescence imaging has shown that the recovery of the photosynthetic activity started from the parts adjacent to the veins and gradually extended to the interveinal parts (Solti et al. 2008) indicating clearly that Cd interfered with Fe uptake and/or transportation in the plants. Therefore Fe-deficiency can be considered as a key factor in Cd-induced inhibition of photosynthesis.

The molecular basis of Cd-induced Fe-deficiency in some plants is that phytosiderophores and other complexing agents excreted by plants can also chelate other metals including Cd and Pb (Strategy II plants; reviewed by Kochian 1995, Hill et al. 2002, Fodor 2006). In Strategy I plants (dicots and nongraminaceous monocots), metallic pollutants interfere with root ferric-chelate reduction (Cd, Pb, Cu, Ni, Mo, Zn) (reviewed by Fodor 2006). However, in some cases, there is no clear correlation between the Fe content of the leaves of metal-treated plants and the decrease in various physiological parameters, suggesting that the total Fe content in the tissues is not neces-sarily the same one as the active Fe pool (Sárvári et al. 1999, reviewed by Fodor 2002). Cd treat-ment also induced decreased levels of micronutrients (Cu, Zn, Fe, Mn, Mo, Ni) and macronutrients (Ca, K, Mg, N, P, S) in leaves and roots (reviewed by Fodor 2002, Pál et al. 2006, Chen et al. 2009, Hasan et al. 2009). This process might be related to the fact that Cd was shown to decrease water uptake and thereby the amount of all transported nutrients (cucumber roots: Varga et al. 1999). The Cd-induced Ca-deficiency in pea plants caused the downregulation of Cu/Zn-SOD, leading to the


overproduction of the ROS hydrogen peroxide and superoxide, as monitored in vivo by confocal laser microscopy (Rodríguez-Serrano et al. 2009). The production of these ROS was mainly associ-ated with vascular tissue, epidermis, and mesophyll cells, and the production of superoxide radicals could be prevented by exogenous Ca (Rodríguez-Serrano et al. 2009). The toxic effects of Cd were reduced in the presence of elevated amounts of Ca in runner bean plants (Skórzyńska-Polit et al. 1998) and in transgenic tobacco plants transformed with TaLCT1 (Antosiewicz and Hennig 2004). Addition of Ca to lettuce plants increased tolerance and accumulation of Cd, while La decreased Cd accumulation (reviewed by He et al. 2005). Interestingly, other authors found no effect of Cd on Ca accumulation in rice (Cui et al. 2008) or found positive effect on Ca accumulation in sugar beet (Larbi et al. 2002). Some experiments with lettuce have shown that increasing Cd concentrations increased the Mn uptake (Ramos et al. 2002), while in cucumber (Sárvári et al. 1999) and tomato (Baszyński et al. 1980) the opposite tendency was found. Addition of Mn to the nutrient solution of hydroponically grown Cd-treated tomatoes restored the photosynthetic pigment content and the photosynthetic activity of the seedlings and induced grana formation (Baszyński et al. 1980).

Similarly to Cd, Pb was also shown to cause Fe-deficiency in plants (Wallace et al. 1992, Varga et al. 2002, Sinha et al. 2006) and chlorosis (Sinha et al. 2006, reviewed by Fodor 2002). Pb had an inhibitory effect on Fe-chelate reductase activity in sugar beet (Larbi et al. 2002). In this study, Pb had only a minor effect on other nutrient concentrations. Pb contamination leads to a drastic reduction of Ca accumulation in cucumber roots and a slight increment in the transport of the essential nutrients, especially Mn (Varga et al. 1999, Cseh et al. 2000). In different experiments, Pb decreased Ca, Mn, K, Zn (reviewed by Fodor 2002), and Ni (reviewed by Chen et al. 2009) contents of the plants. In cabbage, Pb decreased the Mn and Cu contents (Sinha et al. 2006). Cr3+ induces Fe-deficiency in cabbage (Pandey and Sharma 2003). Interestingly, in Cr6+-treated bean seedlings, higher Cr concentrations, decreased Chl content, and drastic changes in chloroplast structure were observed, while the plastid ultrastructure and Chl content of primary leaves that accumulated less Cr was almost not affected (Vázquez et al. 1987). Exposure of spruce plants to both organic and inorganic Hg resulted in a loss of K, Mg, and Mn and accumulation of Fe, indicating that essential-nutrient deficiency may also contribute to the toxic physiological effects observed under excess Hg (for a review see Boening 2000).

26.3.3 SOME UNUSUAL PHENOMENA ASSOCIATED WITH HEAVY-METAL STRESS

In the literature related to HM stress, there are some data that are not often discussed in metal-stress related reviews. In some cases, the excess of one metal has a positive effect on the uptake of another one. On the other hand, low concentrations of HMs sometimes had a positive effect on plant metabolism. In this section, we briefly summarize these phenomena.

26.3.3.1 When the Excess of a Metal Alleviates the Stress Caused by Another MetalAs stated and illustrated above (Table 26.1), the inhibitory effect of excess metals on plant metabo-lism are very often related to the fact that they compete for uptake and transportation and induce deficiency in essential nutrients. This is also true for unbalanced concentrations of essential HMs present in the soil, but it is more evident for toxic elements. When the amount of the toxic metal is low, its uptake into the cells or organs is probably rather weak because its chance to be taken up and to be transported is relatively low. The situation may dramatically change when the amount of toxic metal(s) increases in the environment. Additionally, changes in the concentration may also affect the amount of metals passively transported. Therefore, it is not surprising that in several of these cases, the toxic effects and symptoms induced by the metal in excess can be alleviated by the addition of the essential metal with which it is in competition (reviewed by Poirier et al. 2008). This way, the excess metal-induced deficiency, and the disturbed essential metal homeostasis can be recovered. In these cases, the presence of a beneficial element plays an antidote role against the non-beneficial element, even when it is given in excess.


Several experiments have shown that the Fe nutrition status of plants may significantly modify nonessential metal uptake (reviewed by Fodor 2006). Experiments on the interaction of Fe supply with Cd uptake revealed that an overdose of Fe decreases Cd accumulation in chloroplasts of pri-mary bean leaves treated with Cd via the nutrient solution (Siedlecka and Krupa 1996). Similarly, the addition of excess Cu decreased the Cd uptake of rice plants (Cui et al. 2008). A moderate excess of Fe results in increased growth and photosynthetic pigment content and more efficient light phase of photosynthesis in Cd-treated plants as a result of the recovery from Cd-induced physiological Fe-deficiency (Solti et al. 2008, for reviews, see Siedlecka and Krupa 1999, Fodor 2006).

Cd-induced decreased photosynthetic rate, carotenoid and Chl contents, as well as disturbed plastid ultrastructure could be partially recovered by the supplementation of Mn in the growth media; at the same time the Cd content of the different plant organs (leaves, stems, and roots) decreased (Baszyński et al. 1980).

Surprisingly, there are also results indicating the positive effect of toxic or nonessential HMs on the uptake of essential metals; however, the exact molecular background of these observations is not completely clear. Some experiments with lettuce have shown that increasing Cd concentrations increased the Mn uptake (Ramos et al. 2002). Pb contamination of cucumber leads to a slight incre-ment in the transport of the essential nutrients, especially Mn (Varga et al. 1999, Cseh et al. 2000). Increasing Pb concentrations increased the Zn content in cabbage leaves (Sinha et al. 2006). Low concentrations of Cr3+ restored the chloroplast ultrastructure in Fe-deficient common bean plants (Poschenrieder et al. 1991).

Silicon is known to effectively mitigate various abiotic stresses such as Cd, Mn, and Al pollution, and also salinity, drought, chilling, and freezing. However, mechanisms of Si-mediated alleviation of abiotic stresses remain poorly understood. The key mechanisms of Si-mediated alleviation of abiotic stresses in higher plants include (1) stimulation of antioxidant systems in plants, (2) com-plexation or coprecipitation of toxic metal ions with Si, (3) immobilization of toxic metal ions in growth media, (4) uptake processes, and (5) compartmentation of metal ions within plants (reviewed by Liang et al. 2007). In rice, the addition of Si to nutrient solutions had a positive effect on plant growth and could decrease the Cd accumulation in the shoots and had therefore an enhancing effect on shoot and root biomass under moderate Cd stress (Zhang et al. 2008).

26.3.3.2 When Nonessential Metals Added at Low Concentrations Have a Stimulating Effect

The toxic and damaging effects of metals are usually observed when the stressors are applied at relatively high concentrations (10−5–10−3 M). Nevertheless, these harmful compounds used at low concentrations (defined as “low-concentration stressors,” 10−8 –10−6 M) may have a beneficial effect on plants. However, the effect depends on the developmental stage of the plant at the beginning of the treatment. The low-concentration stressors stimulate biosynthetic processes and growth in seedlings, and delay aging in detached leaves, which is reflected also by an increased metabolism (Nyitrai et al. 2007, 2009, Kovács et al. 2009). The stimulating effect is nonspecific, i.e., indepen-dent of the agent used (Nyitrai et al. 2003, 2004).

The acceleration of plant growth is one of the stimulatory effects of nonessential metals observed in these studies. Short-term Cd treatments of maize roots result in small growth stimu-lation (Wójcik and Tukendorf 1999). Similarly, long-term treatments with Pb, Ni, and Ti induce a significant increase in root length, dry and fresh weights, and shoot growth in maize and bean (Nyitrai et al. 2003).

Low-concentration stressors often stimulate the metabolic activity, i.e., respiration, Chl biosyn-thesis, and photosynthetic activity in seedlings (Prasad et al. 2001, Nyitrai et al. 2004, Nyitrai et al. 2007). At the germination stage, Cd and Pb significantly increase the levels of Chls and carotenoids until the fourth day of development (Shaw and Rout 1998). Similarly, at relatively low concentrations, Pb has a slight stimulatory effect on the Chl content of cucumber leaves (Sárvári et al. 1999). Cd treatment of young maize seedlings increases the leaf Chl (Drazkiewicz et al. 2003,


Drazkiewicz and Baszyński 2005) and carotenoid (Drazkiewicz and Baszyński 2005) content up to 100 μM, but causes chlorosis when applied above this concentration. Low concentrations of Cr3+ enhance the growth of bean plants and reduce the chlorosis in young leaves of Fe-deficient plants by increasing the concentration of Chls and carotenoids (Bonet et al. 1991, Poschenrieder et al. 1991).

One of the most comprehensive surveys in this field is that of Nyitrai et al. (2003). In this study, low concentrations of different metals (Cd: 5 × 10−8 and 10−7 M, Pb and Ni: 10−7 and 10−6 M, Ti: 10−6 and 10−5 M) had a stimulating effect on Chl biosynthesis and photosynthetic activity of bean and maize. Treatments applied either in the nutrient solutions or by spraying the leaves were both effec-tive. However, the extent of the stimulating effect depended on the species, the time course of treat-ment, the position of leaf, and the agent (Nyitrai et al. 2003). The stimulation of the photosynthetic activity was observed at different intervals during all treatments (Cd, Pb, Ni, Ti), while Chl a/b ratios of leaves or chloroplasts did not change considerably (Nyitrai et al. 2003). Low-concentration stressors increased the amount of PSI and LHCII. Electron microscopy of either maize or bean leaves did not show significant differences in the chloroplast lamellar systems between control and treated plants. The authors concluded that these low-concentration stressors generate nonspecific alarm reactions in plants, which may involve changes of the hormonal (e.g., cytokinin) balance (Nyitrai et al. 2003, 2004). In a later study, the authors have demonstrated that in barley seed-lings treated with low concentrations of Cd (5 × 10−8 M), the amount of cytokinins increases in the roots, and it is transported to the leaves where it also causes stimulation (Kovács et al. 2009). The phosphatidylinositol-4,5-biphosphate-inositol-1,4,5-triphosphate/diacylglycerol and the mitogen-activated protein kinase signaling pathways, and not the stressor itself, were found to be responsible for the primary stimulation of cytokinin synthesis and/or activation in the roots (Kovács et al. 2009). Cd treatment at this low concentration did not induce any oxidative stress either in the roots or in the leaves, where Cd did not even accumulate to detectable amounts.

In model systems (i.e., the detached old leaves of different plants), the beneficial effect of the mod-erate and weak stress that induces nonspecific alarm responses in plants could be studied and was shown to have a rejuvenating effect on plastids and their metabolism (Nyitrai et al. 2007, 2009). The physiological parameters of detached leaves are stimulated in a similar way to those observed in the case of seedlings (Nyitrai et al. 2003, Kovács et al. 2009). At the same time, the stressors stimulate starch accumulation in the chloroplasts, and a decrease of the large plastoglobuli typical for plastid senescence (Nyitrai et al. 2004, 2007). Under Pb (10−7 M) and Ti (10−6 M) treatment of detached, non-rooting barley leaves, the level of active cytokinins is not affected, indicating the direct effect of the stressors in this experimental system (Nyitrai et al. 2007). In both model systems (detached bean and barley leaves) the phosphatidylinositol-4,5-biphosphate-inositol-1,4,5-triphosphate/diacylglycerol sig-naling pathway is involved in the anti-senescence effect (Nyitrai et al. 2007, 2009). These interesting data provide further evidences for the complexity of interactions of plastids and metals.

26.4 CONCLUSION

In case of metal pollution in the environment, an unbalanced metal content is often observed in plastids: potentially toxic HMs are present and essential elements are missing. Thanks to various strategies, the cells can cope with a moderate unbalance, but in case of strong unbalance, too many essential biomolecules are not functional any more, photosynthesis is altered and plant productiv-ity is decreased. Although sufficient data are available on HM transporters, it appears that further studies on the mechanism of HM uptake and regulation in plastids are needed. Also, the metal translocation in the whole plant cell, and especially the transporters of the tonoplast that favor the elimination of toxic elements should be further studied. Moreover, the visualization of metal move-ment and transport at the cellular scale would greatly help. A possibility of preventing the entrance of the nonessential elements in plastids would be to produce mutants with more metal-specific transporters. The mechanisms allowing non-essential metals to have a stimulating effect on plant growth should be also further investigated.


ABBREVIATIONS

ATP Adenosine triphosphateChl ChlorophyllDGDG Digalactosyl-diacylglycerolDNA Deoxyribonucleic acidHM Heavy metalHMA HM ATPaseLHC Light-harvesting complexMGDG Monogalactosyl-diacylglycerolNADP+/NADPH Nicotinamide adenine dinucleotide phosphate (oxidized/reduced form)OEC Oxygen-evolving complexPS PhotosystemRNA Ribonucleic acidROS Reactive oxygen speciesRUBISCO Ribulose bisphosphate carboxylase oxygenaseSOD Superoxide dismutase

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Soil metals, chloroplasts, and secure crop production: a review