Heavy Metals and Plants - a complicated relationship · Heavy Metals and Plants - a complicated...

Heavy Metals and Plants - a complicated relationshipHeavy metal toxicity and detoxification

Schwermetall-Hyperakkumulation im Wilden Westenmodified from:

Dose-Response principle for heavy metals

inhi

bito

ry

Effe

ct

stim

ulat

ory

Concentration

Exclusively toxic metal (e.g. mercury) Micronutrient (e.g. copper)

Part I: Heavy metal toxicity

A seemingly intact, natural creek ...However, the Elodea canadensisinside died from zinc stress that converted its chlorophyll to Zn-chlorophyll

Environmental relevance of heavy metal toxicity

Küpper H, Küpper F, Spiller M (1996) Journal of Experimental Botany 47 (295), 259-66

Zn-Fluosilicate

1. Inhibition of root function

Why roots?

• In terrestrial plants the root is generelly the first organ that comes into contact with the heavy metals.

• In the case of heavy metals with typically low mobility, e.g. copper, also the highest metal accumulation is found in the roots

Inhibition of root function

Mechanisms

• Competition in the uptake of less available essential micronutrients, which are sometimes transported by the same proteins

• Enhanced precipitation of essential micronutrients at the root surface

• Inhibition of transport proteins?

• Diverse relatively unspecific inhibitions of cytoplasmic enzymes

• Inhibition of cells division (relevance and mechanism unclear!)

• As a result of roots toxicity, roots tips and root hairs die off

From: Küpper H, Kochian LV, 2008,

unpublished

2. GenotoxicityRelevance

Strongly DEPENDS on the metal applied:

• NOT relevant for copper and zinc toxicity, because other mechanisms (mainly photosynthesis inhibition) are MUCH more efficient

• VERY relavant for cadmium, because genotoxicity is very efficient, comparable e.g. to photosynthesis inhibition

• For lead, it is not very efficient, but other mechanisms are even less efficient because the metal is generally NOT very toxic for plants! Pb toxicity in general NOT environmentally relevant !

Also depends on the plant species!

From: Steinkellner H, et al., 1998, Env.Mol.Mutag. 31, 183-191

Micronucleus (MCN) formation

Genotoxicity

Mechanisms: Point Mutations and Homologous recombinations

From: Kovalchuk O, Titov V, Hohn B, Kovalchuk I, 2001, Nature Biotechnol. 19, 568-72

Hom

olog

ous

reco

mbi

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ns: s

pots

per

pla

nt

Poin

t mut

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ns: s

pots

per

pla

nt

Genotoxicity

Mechanisms: Mitotic aberrations induced by phenyl mercuric acetate (PMA)

From: Dash S, Panda KK, Panda BB, 1988, Mutation Research 203, 11-21

Micronucleus (MCN) formation

C-metaphase Tripolar anaphase

Star anaphaseAnaphase with a pair of lagging chromosomes

Micronucleus (MCN) formationAbnormal spindle

PMA concentration (ppm) PMA concentration (ppm)

3. Oxidative Stress

Mechanisms generating reactive oxygen species during heavy metal stress

• Direct: catalysed by redox-active metal ions (Fe2+, Cu+), hydrogen peroxide is converted to reactive oxygen radicals via the Fenton-Reaction:

• Indirect: malfunktion of photosynthesis and respiration can generate reactive oxygen species. Therefore, even in vivo redox-inert metal ions like Zn2+ and Cd2+ can cause oxidative stress.

Relevance

• NOT clear: Studies with environmentally relevant realistic but still toxic heavy metal concentrations oft do NOT show oxidative stress! Almost all studies concluding that oxidative stress would be a major factor in heavy metal induced inhibition of plant metabolism were carried out using extremely high metal concentrations.

Oxidative Stress

Mechanisms of damage caused by oxidative stress in plants

•Oxidative stress can lead to oxidation of lipids in membranes and thus make them leaky.This is a popular but debated mechanism.

• Oxidation of proteins

From: en.wikipedia.org

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fluorescence


Environmental relevance of heavy metal induced inhibition of photosynthesis:

Elodea stressed by 0.2 µM (= 0.013 ppm) Cu2+

2 H2O O2 + 4 H+

P700*

P700

A0 A1

4. Targets of heavy-metal induced damageto the photosynthetic apparatus

P680*

P680

PheoQA QB

WSC 4 e-

EChlChlPQ FeS/

Rieske

PC

electron transport

Antenna Chl-proteincomplexes,

main protein:LHCII4 h·ν

excitation energy transfer

ChlChl

Chl

heavy metals

Macroscopically visible symptoms of heavy metal damageto photosynthetic systems

Sun reactionIn high irradiance, only a small fraction of the total Chl is accessible to heavy metal Chl formation, and direct damage to the PS II core occurs instead. The bulk of the pigments bleaches, in parallel to the destruction of the photosynthetic apparatus.

Shade ReactionUnder low irradiance conditions that include a dark phase, the majority of antenna (LHC II) chlorophylls is accessible to heavy metal Chlformation by substitution of the natural central ion of Chl, Mg2+. If stable heavy metal Chls (e.g. Cu-Chl) are formed, plants remain green even when they are dead.

Küpper H, Küpper F, Spiller M (1998) Photosynthesis Research 58, 125-33

Why are heavy metal chlorophylls unsuitable for photosynthesis?

• shift of absorbance/fluorescence bands --> less energy transfer

• unstable singlet excited state --> “black holes“ for excitons

• different structure --> proteins denature

• do not readily perform charge separation when in reaction centre.

UV/VIS absorbance spectroscopySpectra of Elodea canadensis extracts


In vivo UV/VIS absorbance spectroscopy


Brown algaEctocarpus siliculosus:

Chl a/c-LHC always accessible to Mg-substitution

--> always shade reaction

Red algaAntithamnion plumula:Phycobilisomes: no Chl--> always sun reaction

Küpper H, Šetlík I, Spiller M, Küpper FC, Prášil O (2002) Journal of Phycology 38(3), 429-441

UV/VIS -spectroscopy: non-Chlorophyta

Photosynthesis activity: Sun- vs. Shade-reaction

0

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Mg-subst.: % of control

x10

Fv / F

m:

% of controlGPOR:

% of control

shade reaction sun reaction

Fm

: % of control


0 1 2 3 4 5 6 7 8 9 10 11 12

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fluorescence


Comparison of gas exchange and steady state fluorescence in Elodea stressed by 0.2 µM Cu2+ during shade reaction

Static fluorescence microscopy of metal-stressed Elodea

red (650-700nm) chlorophyll fluorescence

transmittant light observation

1 µM Cu2+ control 100 µM Zn2+


Inhibition of PSI vs. PSII


In vivo thermoluminescence measurements

--> Heavy metal stress does not lead to a shift in the QA band of thermoluminescence, but only to a lowering of amplitude, i.e. a decrease of the number of active PSII--> QA site is not the primary site of

heavy metal action under physiological conditions


Cd-acclimation in the Zn-/Cd-hyperaccumulator T. caerulescens

Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol., 10.1111/j.1469-8137.2007.02139.x.

0.00.10.20.30.40.50.60.70.8

dark acclimated light acclimated at the end of the recovery period

control palisade mesophyll

F v/ F

m

; Φ

PSII =

(Fm'-F

t')/ F

m' spongy mesophyll

0.00.10.20.30.40.50.60.70.8

NP

Q =

(Fm-F

m')/

Fm

Mrz Mai Jul Sep Nov0.00.10.20.30.40.50.60.70.8

Sat

urat

ion

= (F

p-F 0

)/(F m

-F0)

DateMrz Mai Jul Sep Nov

10 µM Cd2+

palisade mesophyll spongy mesophyll

Mrz Mai Jul Sep NovDate

Mrz Mai Jul Sep Nov

Cd-stress in the Zn-/Cd-hyperaccumulator T. caerulescens: Spectral changes of PSII activity parameters


NIR-luminescence study of excitation energy transfer between chlorophyll derivatives and singlet oxygen

020406080

100120140160180200

Chl a derivatives Chl b derivativeseffi

cien

cy o

f sin

glet

oxy

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of M

g-C

hl a

Mg2+ H+ (=pheophytin) Cu2+ Zn2+

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hl a

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Chl a derivatives Chl b derivatives

Mg2+ H+ (=pheophytin) Cu2+ Zn2+

lifet

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of s

ingl

et o

xyge

n/ %

of M

g-C

hl a

--> Hms-Chls have lower or equal quantum yields of singlet oxygen (1O2) production, but always lower yields of 1O2quenching compared to Mg-Chl. Phe has the most efficient 1O2 production and least efficient quenching. --> Hms-Chl formation may indirectly lead to oxidative stress.

Küpper H, Dedic R, Svoboda A, Hála J, Kroneck PMH (2002) Biochim Biophys Act 1572, 107-113

Part II: Heavy detoxification

1. Heavy metal detoxification with strong ligands

Phytochelatins

• Bind Cd2+ with very high affinity, but other heavy metal ions with low affinity

• Specially for Cd2+-binding synthesized by phytochelatin-synthase

• They are the main Cd-resistance mechanism in most plants (excepthyperaccumulators) and many animals

• Phytochelatin synthase becomes activated by (e.g. via Cd-binding) blocked thiols ofglutathion and similar peptides

• Phytochelatins bind Cd2+ in the cytoplasm, then the complex is sequestered in the vacuole.

• In the vacuole large phytochelatin-Cd-aggregates are formed

Metallothionins

• MTs of type I und II bind Cu+ with high affinity andseem to be involved in its detoxification.

• BUT: Main role of MTs in plants seems to be metal-distribution during the normal (non-stressed)metabolism

Glutathion

• Also glutathione itself, the building block of phytochelatins, can bind and thus detoxify heavy metals - the in vivo relevance is questionable


Free Amino acids

• Like during metal transport under normal physiological conditions, free amino acids(mainly histidine+nicotianamine) seem to play a role in the detoxification of heavy metals.

Other Ligands

• Diverse Proteins

• Anthocyanins: seem to be involved in Brassicacae in molybdemum binding(detoxification or storage?)

• Cell wall

• Some algae release unidentified thiol-ligands during Cu-stress


NicotianamineHistidine

Speciation of cadmium and copper in the Cu-sensitive CdZn-hyperaccumulator T. caerulescens

Analysed by XAS of frozen-hydrated tissues

Cd: Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2003) Plant Physiology 134 (2), 748-757Cu: Mijovilovich A, Meyer-Klaucke W, Kroneck PMH, Küpper H - unpublished

Cu-K-edge EXAFS

2. Resistance mechanisms against oxidative stress

• Enhanced expression of enzymes that detoxify reactive oxygen species (superoxide dismutase+catalase. Problem: inhibition of Zn-uptake ( SOD) during Cd-Stress.

• Synthesis of non-enzyme-antioxidants, e.g. ascorbate and glutathione

• Changes in the cell membranes to make them more resistant against the attack ofreactive oxygen species:- Lipids with less unsaturated bonds- Exchange of phosphatidyl-choline against phosphytidyl-ethanolamine as lipid-“head“- Diminished proportion of lipids and enhanced proportion of stabilising proteins in themembrane

3. Heavy metal detoxification by compartmentation

Mechanisms

• Generelly: aktive transport processes against the concentration gradienttransport proteins involved.

• Exclusion from cells:- observed in brown algae- in roots

• Sequestration in the vacuole: - plant-specific mechanism (animals+bacteria usually don‘t have vacuoles...)- very efficient, because the vacuole does not contain sensitive enzymes- saves the investment into the synthesis of strong ligands like phytochelatins- main mechanism in hyperaccumulators

• Sequestration in least sensitive tissues, e.g. the epidermis instead of the photosynthetically active mesophyll

Compartmentation of metals in leavesZn/Cd/Ni accumulation in epidermal vacuoles

of Thlaspi and Alyssum species

Zn Mg P S Cl K0

2

4

6

8mature leaves

mM

/ mM

Ca

Zn Mg P S Cl K0

2

4

6

8 young leaves

mM

/ mM

Ca

upper epidermis upper mesophylllower mesophyll lower epidermis

Concentrations of elements in leaf tissues Thlaspi caerulescens

Zn K α line scan and dot map of a T. caerulescens leaf

Ni K α line scan and dot map of a A. bertolonii leaf

Zn: Küpper H, Zhao F, McGrath SP (1999) Plant Physiol 119, 305-11Ni: Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP (2001) J Exp Bot 52 (365), 2291-2300

vacuole

epidermis

mesophyllupper lower

From: Colangelo EP, Guerinot ML, 2006, CurrOpinPlantBiol9:322-330

Mechanisms of Metal Uptake in plantsRoot uptake

example: iron and zinc transport in Brassicaceae

root uptake

intracellular distribution

4 main familiesP-type ATPases (P1B-type = CPX ATPases)Cation Diffusion Facilitators (CDF-transporters)ZRT-/IRT-like proteins (ZIP-transporters)Natural resistance associated macrophage proteins (Nramp-transporters)

From: Williams LE, Mills RF, 2005, TrendsPlantSci10, 491-502

Mechanisms of Metal Uptake+Compartmentation in Plants (I)CPx-type (=P1B-type) ATPases

Likely Functions (suggested by expression studies)

translocation into the root xylem, that means out of root cells ( e.g. HMA4)xylem unloading in shootsintracellular metal sequestration e.g. in

the vacuole (putative!)transport into the chloroplast, inside

the chloroplast into the thylakoidstransport into the Golgi apparatus

From: Argüello JM et al., 2007, Biometals, DOI 10.1007/s10534-006-9055-6

From: Williams LE, Mills RF,2005,

TrendsPlantSci 10,491-502

Mechanisms of Metal Uptake+Compartmentation in Plants (I)CPx-type (=P1B-type) ATPases

Sequence characteristics predicted structure(only A, P, and N-domain crystallised from bacterial proteins)

CPx-motif in 6th predicted transmembrane helix ( Name!)Variable number of transmembrane helicesMANY histidines and cysteines in sequence ( e.g. 58 Cys in TcHMA4)Putative metal binding domain at N-terminus (in cytosol?)Histidine repeat at C-terminus (in cytosol?)

Purification and Characterisation of a Zn/Cd transporting P1Btype ATPase from natural abundance in the Zn/Cd

hyperaccumulator Thlaspi caerulescens

From: Aravind P, Leitenmaier B, Yang M, Welte W, Kochian LV, Kroneck PMH, Küpper H (2007) BBRC 364, 51-56

Preliminary characterisation resultspost-translational modification (splitting) possibleregulation by substrate inhibitionhigher activity with Zn with Cd, but higher affinity for Cd than for Zn

In almost all measured cells, a bright cytoplasmatic ring appeared first after start adding Cd to the medium.

A cell that was incubated with Cdover night is completely filled withCd, which means that the transportinto the vacuole took place

The transport into the vacuole is the time-limiting step in metal uptake!

Leitenmaier B, Küpper H, (2008) unpublished

Cd-transport into protoplasts isolated from the hyperaccumulator plant Thlaspi caerulescens

From:Guerinot ML, 2000, BBA 1465, 190-8

Mechanisms of Metal Uptake+Compartmentation in Plants (II)ZIP-transporters

Structure predicted by sequence• usually 8 transmembrane helices, one long

variable region, predicted to be in the cytoplasm• 309-476 amino acids

Characteristics revelaed by yeast expression studies

• High affinity and saturable kinetics for selected metal (e.g. Zn in ZNT1)

• Lower affinity uptake for related metals (e.g. Cd in ZNT1)

From:Pence NS et al., 2000, PNAS 97, 4956-60

Likely Functions (suggested by expression studies)

• uptake of metals into cells over the cytoplasmatic membrane

• abundant in all eucaryotes, incl. humans, plants and fungi

Fe deficiency (left) and iron replete conditions (right)

From:Connolly et al., 2002, PlantCell 14, 1347-57

Mechanisms of Metal Uptake+Compartmentation in Plants (II)Transcriptional regulation of ZIP transporters

Expression patternexpressed in most plants mainly under metal deficient conditionsin metal hyperaccumulators rather

strong expression at all metal levelsexpression mainly in metabolically

active cells, not metal storage cells

From:Küpper H, Seib LO, Sivaguru M, Hoekenga OA, Kochian LV, 2007 The Plant Journal 50(1), 159-187

Compartmentation of elements in leaves:Correlation between metals in

the mesophyll ofArabidospis halleri

0 2 4 6 8 100

5

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data pointsregression line (R = 0.79; P < 0.0001) 95% confidence limits

Mg

/ mM

Cd / mM0 20 40 60

0

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data points regression line (R = 0.94; P < 0.0001) 95% confidence limits

Cd

/ mM

Zn / mM

Küpper H, Lombi E, Zhao FJ, McGrath SP (2000) Planta 212, 75-84

Cd-stress in the Zn-/Cd-hyperaccumulator T. caerulescens: distribution of photosystem II activity parameters

Cellular Fv/Fm distribution in a control plant

Distribution of Fv/Fm in a Cd-stressed plant


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Proposed mechanism of emergency defenceagainst heavy metal stress

Normal: sequestration in epidermal storage cells

Acclimated: enhancedsequestration in epidermal storage cells

Stressed: additional sequestration in selected mesophyll cells

vvv


4. Root-specific resistance mechanisms

Strategies

• Reduction of the unspecific permeability of the root for unwanted heavy metals:expression of peroxidases enhances lignification

• Active (ATP-dependent) discharge by efflux-pumps ( CPx ATPases!): was shown forCu in Silene vulgaris and for diverse metals in bacteria.

5. Further Resistance-Mechanisms

• Reduction by reductases, e.g. Hg2+ --> Hg0, Cu2+ --> Cu+

• Precipitation of insoluble sulfides outside the cell (on the cell wall)

• Methylation, e.g. of arsenic

Heavy Metals and Plants - a complicated relationship · Heavy Metals and Plants - a complicated...

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