Heavy Metals and Plants - a complicated relationship Heavy metal resistance

l h l i i h ildHeavy metal-hyperaccumulation in the Wild Westmodified from:

Hendrik Küpper, Advanced Course on Bioinorganic Chemistry & Biophysics of Plants, summer semester 2012

Dose-Response principle for heavy metals

Küpper H, Kroneck PMH, 2005, Metal ions Life Sci 2, 31-62

Metal(loid) detoxification: overview of mechanisms proposed for arsenic p p

Briat J PNAS 2010;107:20853-20854

General Resistance-MechanismsH t l d t ifi ti ith t li dHeavy metal detoxification with strong ligands

Phytochelatins (PCs)

• Bind Cd2+ with very high affinity also As(III) and AS(V) but many other heavy metalBind Cd with very high affinity also As(III) and AS(V), but many other heavy metal ions with low affinity

• Specially for Cd2+-binding synthesized by phytochelatin-synthasep y g y y p y y

•PC synthase activated by blocked thiols of glutathion and similar peptides

From: Inouhe M (2011) Braz J Plant Physiol 17, 65-78

General Resistance-MechanismsH t l d t ifi ti ith t li dHeavy metal detoxification with strong ligands

Phytochelatins (PCs)

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

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

Ph h l i Cd f d i l• Phytochelatin-Cd-aggregates are formed in vacuoles

General Resistance-MechanismsH t l d t ifi ti ith t li dHeavy metal detoxification with strong ligands

Glutathion

Also gl tathione itself the b ilding block of ph tochelatins can bind and th s deto if• Also glutathione itself, the building block of phytochelatins, can bind and thus detoxify heavy metals - the in vivo relevance is questionable

Metallothionins

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

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

General Resistance-MechanismsH t l d t ifi ti ith t li d

Other Ligands

N t i i id i ti i ( l i l d i l t t)

Heavy metal detoxification with strong ligands

• Non-proteogenic amino acid nicotianamine (also involved in normal transport)

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

H l t l 2001Hale et al_2001, PlantPhysiol

126, 1391-1402

Cell wall• Cell wall

Carr HP, Lombi E, Küpper H, McGrath SP, Wong MH (2003)

Agronomie 23, 705-10

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

Heavy metal detoxification by compartmentationMechanismsMechanisms

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

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

• Sequestration in the vacuole: Kü H t l 2001 J E B t 52 (365) 2291 2300• Sequestration in the vacuole: - plant-specific mechanism (animals+bacteria usually don‘t have vacuoles...)- very efficient, because the vacuole does not contain sensitive enzymes

Küpper H et al., 2001, J Exp Bot 52 (365), 2291-2300

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

Küpper H, Zhao F, McGrath SP (1999) Plant Physiol 119, 305-11

Mechanisms of Metal transport proteins

∆G= nIonen * R * T * ln (cinside / coutside) + 3F (φoutside-φinside)(R = gas constant, T = temperature, F = Faraday constant, φ = electrochemical potential)

Further Resistance-Mechanisms

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

R h CL t l 1996 PNAS 93Rugh CL, et al, 1996, PNAS 93, 3182-3187

P i it ti f i l bl lfid t id th ll ( th ll ll)• Precipitation of insoluble sulfides outside the cell (on the cell wall)

• Methylation, e.g. of arsenic

Root-specific resistance mechanismsZn-sensitive

Strategies

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

• Active (ATP-dependent) discharge by efflux-pumps: was shown for Cu in Silene vulgaris(and for diverse metals in bacteria) Zn-resistant(and for diverse metals in bacteria).

Chardonnens AN, Koevoets PLM, vanZanten A, Schat H, Verkleij JAC, 1999, PlantPhysiol120_779-785

Purification and Characterisation of the Zn/Cd transporting P1B type ATPase from the p g 1B yp

Zn/Cd hyperaccumulator T. caerulescens

Scheme from: Solioz M, Vulpe C 1996) TIBS21_237-41

TcHMA4 protein is smaller than predicted by cDNA post-translational processing

Maximal pumping activty of TcHMA4 at similar concentrations as e.g. ATP7b from humansfrom humans

At higher, but still physiological concentrations:physiological concentrations: inactivation and/or change of pumping direction

Barbara Leitenmaier, Annelie Witt, Annabell Witzke, Anastasia Stemke, Wolfram Meyer-Klaucke ,Peter M.H. Kroneck, Hendrik Küpper (2011) Biochimica et Biophysica Acta (Biomembranes) 1808, 2591-2599

Resistance mechanisms against oxidative stress

• Enhanced expression of enzymes that detoxify reactive oxygen species (superoxideEnhanced 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

Ch i th ll b t k th i t t i t th tt k f• Changes in the cell membranes to make them more resistant against the attack of reactive 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 the membrane

Heavy metal uptake characteristics of plants

Review: Küpper H, Kroneck PMH, 2005, Metal ions Life Sci 2, 31-62

Plants with an unusual appetite:Heavy metal hyperaccumulationy yp

(a)14 Thlaspi goesingense

Alyssum bertolonii

(b)30000

Thlaspi goesingense

ight

(g)

8

10

12 Alyssum bertoloniiAlyssum lesbiacum

on (µ

g g-1

)

20000

25000Thlaspi goesingenseAlyssum bertoloniiAlyssum lesbiacum

hoot

dry

we

4

6

8

conc

entra

tio

10000

15000

Sh

0

2

4

Ni c

0

5000

Ni added to the substrate (mg kg-1)

0 1000 2000 3000 4000

Ni added to the substrate (mg kg-1)

0 1000 2000 3000 4000

Effects of Ni2+ addition on hyperaccumulator plant growth and Ni2+ concentration in shootsplant growth and Ni concentration in shoots

Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP (2001) J Exp Bot 52 (365), 2291-2300

Speciation of cadmium and zinc phyperaccumulated by Thlaspi caerulescens (Ganges ecotype)

revealed by EXAFS of frozen-hydrated tissues

80

g to

Cd

80

g to

Cd

y y

60

s bi

ndin

g

60

s bi

ndin

g

20

40

all l

igan

ds

20

40

all l

igan

d

young mature senescent dead0

20

rcen

t of a

stems petioles leaves0

20

rcen

t of a

young mature senescent dead

Per

Developmental stage of leaves sulphur ligands N/O ligands

stems petioles leaves

Pe

Tissue

Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2004) Plant Physiology 134, 748-757

phytochelatinmetallothionein malic acid histidine

Speciation of hyperaccumulated metals revealed by EXAFS:Cd in the CdZn-hyperaccumulator T. caerulescensyp

and Cu in the Cu-hyperaccumulator C. helmsii

Cd: Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2004) Plant Physiology 134 (2), 748-757Cu: Küpper H, Mijovilovich A, Götz B, Küpper FC, Wolfram Meyer-Klaucke W (2009) Plant Physiol. 151, 702-14

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

Cd: Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2004) Plant Physiology 134 (2), 748-757Cu: Mijovilovich A, Leitenmaier B, Meyer-Klaucke W, Kroneck PMH, Götz B, Küpper H (2009) Plant Physiology

151, 715-31

Speciation of copper in the Cu-sensitive CdZn-hyperaccumulator T caerulescensCu(II)-oxalate structure from Michalowicz et al. (1979) Inorg Chem 18, 3004-310

Fe(III)-Nicotianamine, structure from vonWiren et al. (1999) PlantPhysiol 119

in the Cu-sensitive CdZn-hyperaccumulator T. caerulescensAnalysed by XAS of frozen-hydrated tissues

Cu-oxalate (moolooite)

Cu(I)-metallothioneins & phytochelatinsCu(I) metallothioneins & phytochelatins

O SCu

Cu+C

Cu

O

Cu(II)-NicotianamineCu(II)-aquo and Cu(II)-malate

Cu(I)-MT EXAFS fromSayers et al. (1993) Eur J Biochem 212,

521-528

Cd: Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2003) Plant Physiology 134 (2), 748-757Cu: Mijovilovich A, Leitenmaier B, Meyer-Klaucke W, Kroneck PMH, Götz B, Küpper H (2009) Plant Physiology 151, 715-731

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

of Thlaspi and Alyssum speciesepidermis

6

8 young leaves

mM

Ca vacuole

p

mesophyll

2

4

mM

/ m

upper lower

8mature leaves

Zn Mg P S Cl K0

4

6

M /

mM

Ca

Zn Mg P S Cl K0

2mM

id i h llupper 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 leaftissues Thlaspi caerulescens of a T. caerulescens leaf 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

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

30

T. caerulescens

C 20

T. fendleri

A

0

10

20

Con

trol

0

10

Con

trol

0

10

20 D

esse

d

0

10

20 B

esse

d

Cellular Fv/Fm distribution in a control plant 0

Stre20 E

ng0

Stre

0

10

Accl

imat

in

10

20 F

clim

ated

Distribution of Fv/Fm in a Cd-stressed plant

0.0 0.2 0.4 0.6 0.8 1.00

Fv / Fm

Acc

p

Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytologist 175, 655-674

Proposed mechanism of emergency defenceagainst heavy metal stressagainst heavy metal stress

Acclimated: EnhancedNormal: Sequestration in epidermal storage cells

Acclimated: Enhanced sequestration in epidermal storage cells

Stressed: additional sequestration in selected mesophyll cells

Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytologist 175, 655-674

Kompartimentierung von Metallen in BlätternKorrelation zwischen Metallkonzentrationen

im Mesophyll von Arabidospis halleri

10

20

258

10

15

20

/ mM

4

6

/ mM

5

10

data pointsregression line (R = 0.79; P < 0.0001)

Mg

2

4

data points regression line (R = 0.94; P < 0.0001)

Cd

0 2 4 6 8 100

g ( ; ) 95% confidence limits

Cd / mM0 20 40 60

0 95% confidence limits

Zn / mM Cd / mMZn / mM

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

Mechanisms of Metal Uptake in plantsRoot uptake and intracellular distribution in plantsp p

example: iron and zinc transport in Brassicaceae

root uptake

intracellularintracellular distribution

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

4 main families, all overexpressed in hyperaccumulators! P-type ATPases cation diffusion facilitators (CDF-transporters) cation diffusion facilitators (CDF-transporters) ZRT-/IRT-like proteins (ZIP-transporters) Natural resistance associated Macrophage proteins (Nramp-transporters)

Cd-transport into protoplasts isolated from the hyperaccumulator plant Thlaspi caerulescens... (II)yp p p ( )

In almost all measured cells, a bright cytoplasmatic ring

A cell that was incubated with Cd over night is completely filled with Cd hi h th t th t tappeared first after start adding

Cd to the medium.Cd, which means that the transport into the vacuole took place

Th t t i t th l i th ti li iti t i t l t k !The transport into the vacuole is the time-limiting step in metal uptake!

Leitenmaier B, Küpper H (2011) Plant Cell & Environment 34, 208-219

HMA3 as a likely candidate for the vacuolar Cd sequestration

T.c. Ganges

qin T. caerulescens and

elevated Cd-accumulation in the Ganges vs. Prayon

ecotypeT.c. Prayon

HMA3 is much stronger expressed in T.c. Ganges

HMA3 is localised in the vacuolar membrane

Ueno D et al. et Ma JF, 2011, PlantJ66_852–62

Cd-transport into protoplasts isolated from the hyperaccumulator plant Thlaspi caerulescens...(III)yp p p ( )

higher uptake rates in large metal storage cells compared to other epidermal cells are

caused by higher transporter expression, NOT by

differences in cell walls or transpiration stream

Leitenmaier B, Küpper H (2011) Plant Cell & Environment 34, 208-219

Regulation of ZNT1 transcription analysed by quantitative mRNA in situ hybridisation (QISH)y ( )

in a non-hyperaccumulating and a hyperaccumulating Thlaspi species 10 µM Zn2+ Thlaspi caerulescens10 µM Zn2+ Thlaspi arvense

0.5

10 µM Zn Thlaspi arvense 1 µM Zn2+ Thlaspi arvense

0 3

0.4

(18s

rRN

A)

0.2

0.3

mR

NA

) / c

(

0.1c(ZN

T1

mesop

hyll

phloe

mdle

shea

thmes

ophy

llrag

e cell

sdia

ry ce

llsua

rd ce

lls

0.0

spon

gy m

e

bund

lepa

lisad

e me

rmal

metal s

tora

iderm

al su

bsidi

a ep

iderm

al gu

a

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

epide

rmep

id

Quantitative cellular pattern of ZNT5 transcript abundance in young leaves of Thlaspi carulescens (Ganges ecotype)

judged by its j g yexpression pattern in the epidermis, ZNT5 may be a key player ina key player in hyperaccumulation of Zn

Küpper H, Kochian LV (2010) New Phytologist 185, 114-29

Regulation of ZNT5 transcription in young leaves of Thlaspi carulescens (Ganges ecotype) analysed by QISH

ZNT5 seems to be involved both in unloading Zn from the veins and in sequestering it into epidermal storage cellssequestering it into epidermal storage cells

Küpper H, Kochian LV (2010) New Phytologist 185, 114-29

Purification and Characterisation of a Zn/Cd transporting P1B type ATPase from natural abundance in the Zn/Cd

Scheme from: Solioz M, Vulpe C 1996) TIBS21_237-41

yphyperaccumulator Thlaspi caerulescens

Aravind P, Leitenmaier B, Yang M, Welte W, Kochian LV, Kroneck PMH, Küpper H (2007)

BBRC 364, 51-56

t t l ti l difi ti f T HMA4 post-translational modification of TcHMA4

KD of TcHMA in the high nanomolaar to low micromolar range

EXAFS-analysis of TcHMA4 and another, so far unknown Cd-ATPase

First shell mainly S,

First shell mainly S

mainly S, evidence for Cd-S-cluster from multiple

tt iscattering

Leitenmaier B, Witt A, Witzke A, Stemke A, Meyer-Klaucke W, Kroneck P, Küpper H (2011) accepted for publication in Biochim et Biophys Acta

Activation and substrate inhibition of TcHMA4inhibition of TcHMA4

Leitenmaier B, Witt A, Witzke A, Stemke A, Meyer-Klaucke W, Kroneck P, Küpper H (2011)

accepted for publication in Biochim et Biophys Acta

TcHMA4 Characterisation: 2D-Activity testsTemperature dependence of substrate binding and influence of the substrate on the

thermostability of TcHMA4nM

)Zn

2+))

/ log

(nLo

g (c

(Z

substrate inhibition at high metal

5 50

concentrations!

Temperature / °C5 50

Leitenmaier B, Witt A, Witzke A, Stemke A, Meyer-Klaucke W, Kroneck P, Küpper H (2011) accepted for publication in Biochim et Biophys Acta

All slides of my lectures can be downloadedAll slides of my lectures can be downloaded

from my workgroup homepagefrom my workgroup homepage www.uni-konstanz.de Department of Biology Workgroups Küpper lab,

or directlyhttp://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_Homepage.html

and

on the ILIAS website