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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
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Dose-Response principle for heavy metals
Küpper H, Kroneck PMH, 2005, Metal ions Life Sci 2, 31-62
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Metal(loid) detoxification: overview of mechanisms proposed for arsenic p p
Briat J PNAS 2010;107:20853-20854
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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
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Heavy metal uptake characteristics of plants
Review: Küpper H, Kroneck PMH, 2005, Metal ions Life Sci 2, 31-62
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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