Heavy Metals and Plants - a complicated relationshipHeavy metal detoxification
Schwermetall-Hyperakkumulation im Wilden Westenmodified from:
Presented by Hendrik Küpper, Universität Konstanz, at the DAAD summer school 2008
Dose-Response principle for heavy metals
Küpper H, Kroneck PMH, 2005, Metal ions Life Sci 2, 31-62
Cd-acclimation in Thlaspi caerulescens
Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74
1. General Resistance-Mechanisms1.1. 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 activvated 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
1. General Resistance-Mechanisms1.1. Heavy metal detoxification with strong ligands
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.
1. General Resistance-Mechanisms1.1. Heavy metal detoxification with strong ligands
Nicotianamine
Histidine
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
1. General Resistance-Mechanisms1.1. Heavy metal detoxification with strong ligands
Carr HP, Lombi E, Küpper H, McGrath SP, Wong MH (2003)
Agronomie 23, 705-10
Hale et al_2001, PlantPhysiol
126, 1391-1402
4.2. Heavy metal detoxification by compartmentationMechanisms
• 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
Küpper H et al., 2001, J Exp Bot 52 (365), 2291-2300
Küpper H, Zhao F, McGrath SP (1999) Plant Physiol 119, 305-11
4.3. 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
Rugh CL, et al, 1996, PNAS 93, 3182-3187
1. 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: was shown for Cu in Silene vulgaris(and for diverse metals in bacteria).
Zn-sensitive
Zn-resistant
Chardonnens AN, Koevoets PLM, vanZanten A, Schat H, Verkleij JAC, 1999, PlantPhysiol120_779-785
3. 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
Part II: Plants with an unusual appetite:Heavy metal hyperaccumulation
(a)
Ni added to the substrate (mg kg-1)
0 1000 2000 3000 4000
Shoo
t dry
wei
ght (
g)
0
2
4
6
8
10
12
14 Thlaspi goesingenseAlyssum bertoloniiAlyssum lesbiacum
Effects of Ni2+ addition on hyperaccumulatorplant growth and Ni2+ concentration in shoots
(b)
Ni added to the substrate (mg kg-1)
0 1000 2000 3000 4000
Ni c
once
ntra
tion
(µg
g-1)
0
5000
10000
15000
20000
25000
30000Thlaspi goesingenseAlyssum bertoloniiAlyssum lesbiacum
Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP (2001) J Exp Bot 52 (365), 2291-2300
Cadmium deficiency in the Cd/Zn hyperaccumulatorThlaspi caerulescens
demonstrates the biological function of hyperaccumulation...
With 10 µM cadmium in the nutrient solution--> healthy plants
Without cadmium in the nutrient solution--> damage due to attack of insects
Küpper H, Kroneck PMH (2004) MIBS 44 (Sigel et al., eds), chapter 5
How do hyperaccumulators bind
heavy metals?
Analysis of metal ligands byExtended X-ray Absorption
Fine Structure (EXAFS)
Speciation of cadmium and zinc hyperaccumulated by Thlaspi caerulescens (Ganges ecotype)
Analysed by X-ray absorption spectroscopy of frozen-hydrated tissues
Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2003) Plant Physiology 134 (2), 748-757
0.0
0.2
0.4
0.6
0.8
1.0
1 2 3 4 50.0
0.2
0.4
0.6
0.8
1.0
young leaves mature leaves mature stems
Zn
histidine contribution
young leaves senescent leaves mature stems
Distance [Å]
Four
ier T
rans
form
of χ*
k3
Cd
increase in sulphur contribution
Speciation of cadmium hyperaccumulated by Thlaspi caerulescens (Ganges ecotype)
Analysed by X-ray absorption spectroscopy of frozen-hydrated tissues
young mature senescent dead0
20
40
60
80
Perc
ent o
f all
ligan
ds b
indi
ng to
Cd
Developmental stage of leaves sulphur ligands N/O ligands
stems petioles leaves0
20
40
60
80
Per
cent
of a
ll lig
ands
bin
ding
to C
d
Tissue
Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2003) Plant Physiology 134 (2), 748-757
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
Analysis: a) recording of complete spectrum, subtraction of background --> quantification of peak areas by comparison to internal standardb) recording of counts in spectral window --> dot maps, line scans
Energy / keV
Cou
nts
continuous backgroundof bremsstrahlung
peaks of characteristic x-ray photons
spectral window
Where do hyperaccumulators store metals ? Measurements by Energy Dispersive X-ray Analysis (EDXA)
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
Compartmentation of metals in shootsSubcellular localisation of Ni in the epidermis of
Thlaspi goesingense
Compartmentation of Zn and Ni in leavesFunctional differenciation of epidermal cells of Thlaspi species
15 20 25 30 35 40 45 500
2
4
6
8
regression lineconfidence limits
data points
Linear Regression: Y = A + B * X
Parameter Value Error-----------------------------------------------A -0.057 1.027B 0.153 0.034-----------------------------------------------
R SD N P-----------------------------------------------0.816 1.079 12 0.00118-----------------------------------------------
mM
Zn
/mM
Ca
cell width
Upper leaf surface of T. goesingense
Dot map of the Ni K alpha lineCorrelation between cell size and zinc concentration
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
Compartmentation of metals in leaves Zn/Cd accumulation in epidermal trichomes of Arabidopsis halleri
Küpper H, Lombi E, Zhao FJ, McGrath SP (2000) Planta 212, 75-84
Compartmentation of elements in leaves:Correlation between metals in
the mesophyll ofArabidospis halleri
0 2 4 6 8 100
5
10
15
20
25
data pointsregression line (R = 0.79; P < 0.0001) 95% confidence limits
Mg
/ mM
Cd / mM0 20 40 60
0
2
4
6
8
10
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
Transmitted light:
information about structureand cell type
Rhod5N fluorescence:
cadmium measurement
Cd-transport into protoplasts isolated from the hyperaccumulator plant Thlaspi caerulescens revealed by
in vivo fluorescence kinetic microscopy
Leitenmaier B, Küpper H, (2008) unpublished
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... (II)
Leitenmaier B, Küpper H, (2008) unpublished
Cd-transport into protoplasts isolated from the hyperaccumulator plant Thlaspi caerulescens...(III)
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
Regulation of ZNT1 transcription analysed by quantitative mRNA in situ hybridisation (QISH)
in a non-hyperaccumulating and a hyperaccumulating Thlaspi species
Küpper H, Seib LO, Sivaguru M, Hoekenga OA, Kochian LV (2007) The Plant Journal 50(1), 159-187
spon
gy m
esop
hyll
phloe
mbu
ndle
shea
thpa
lisad
e mes
ophy
ll
epide
rmal
metal s
torag
e cell
s
epide
rmal
subs
idiary
cells
epide
rmal
guard
cells
0.0
0.1
0.2
0.3
0.4
0.5
c(ZN
T1 m
RN
A) /
c(1
8s rR
NA
) 10 µM Zn2+ Thlaspi caerulescens 10 µM Zn2+ Thlaspi arvense 1 µM Zn2+ Thlaspi arvense
Quantitative cellular pattern of ZNT5 transcript abundance in young leaves of Thlaspi carulescens (Ganges ecotype)
Küpper H, Seib LO, Kochian LV - unpublished
judged by its expression pattern in the epidermis, ZNT5 may be a key player in hyperaccumulation of Zn
Regulation of ZNT5 transcription in young leaves of Thlaspi carulescens (Ganges ecotype) analysed by QISH
Küpper H, Seib LO, Kochian LV - unpublished
ZNT5 seems to be involved both in unloading Zn from the veins and in sequestering it into epidermal storage cells
Scheme from: Solioz M, Vulpe C 1996) TIBS21_237-41
Purification and Characterisation of a Zn/Cd transporting P1B type ATPase from natural abundance in the Zn/Cd
hyperaccumulator Thlaspi caerulescens
Aravind P, Leitenmaier B, Yang M, Welte W, KochianLV, Kroneck PMH, Küpper H (2007) BBRC 364, 51-56
post-translational modification of TcHMA4
KD of TcHMA in the high nanomolaar to low micromollar range
UV- Vis-Spectroscopy of TcHMA4
Ligand-metal charge-transfer transitions of Cd-cysteine bonds
Leitenmaier B, Meyer-Klaucke W, Aravind P, Kroneck PMH, Küpper H (2008) unpublished
EXAFS-analysis of TcHMA4 and another, so far unknown Cd-ATPase
First shellmainly S
First shellmainly S, evidence forCd-S-clusterfrom multiple scattering
Leitenmaier B, Meyer-Klaucke W, Aravind P, Kroneck PMH, Küpper H (2008) unpublished
Compartmentation of elements in leaves:Correlation between metals and cells with differing physiology in
the mesophyll of metal-stressed hyperaccumulators
0 2 4 6 8 100
5
10
15
20
25
data pointsregression line (R = 0.79; P < 0.0001) 95% confidence limits
Mg
/ mM
Cd / mM0 20 40 60
0
2
4
6
8
10
data points regression line (R = 0.94; P < 0.0001) 95% confidence limits
Cd
/ mM
Zn / mMCd vs. Zn, Cd vs. Mg: Küpper H, Lombi E, Zhao FJ, McGrath SP (2000) Planta 212, 75-84
Ni vs. Mg: Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP (2001) J Exp Bot 52 (365), 2291-2300Heavy metal chlorophylls: Küpper H et al. 1996 JExpBot, 1998 PhotRes, 2002 JPhycol, 2003 FunctPlantBiol
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
0
10
20
30
T. caerulescens
C
Con
trol
0
10
20 D
Stre
ssed
0
10
20 EAc
clim
atin
g
0.0 0.2 0.4 0.6 0.8 1.00
10
20 F
Fv / Fm
Acc
limat
ed
0
10
20
T. fendleri
Con
trol
A
0
10
20 B
Str
esse
d
Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74
transient heterogeneity of mesophyll activity during period of Cd-induced stress
Cd-stress in the Zn-/Cd-hyperaccumulator T. caerulescens:correlation between PSII activity parameters and Cd-accumulation
Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74
Fv/Fm Fluorescence of Cd-dye
transient heterogeneity of mesophyll activity during period of Cd-induced stress
correlates with transient heterogeneity of Cd-accumulation!
Proposed mechanism of emergency defenceagainst heavy metal stress
Normal: Sequestration in epidermal storage cells
Acclimated: Enhanced sequestration in epidermal storage cells
Stressed: additional sequestration in selected mesophyll cells
vvv
Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74
plant species max. Cd Biomass Cd-removalmg/kg DW t DW/ha g/(ha*year)
Arabidopsis halleri 100 2 200Thlaspi caerulescens (Prayon) 250 5 1250Thlaspi caerulescens (S. France)2500 5 12500Dichapetalum gelonoides 2.1 5 10Athyrium yokosense 165 2 330Arenaria patula 238 2 476Sedum alfredii 180 5 900Poplar/Pappel 2.5 20 50Upland Rice 40. 10 400
Data from field experiments, carried out by Rufus Chaney (USA), presented in Hangzhou 2005
Phytoremediation – comparison of species
hyperaccumulators are, despite their low biomass, the best-suited plants for phytoremediation!
Vegetation on naturally nickel-rich soil(Serpentine). Such soil is neither usable for agriculture (Ni-concentration far too high) nor for conventional ore mining (Ni-concentration too low).
Application of hyperaccumulators fro phytomining
Nickel-hyperaccumulators on such soils enrich the Ni to several percent of their shoot dry mass. After burning them, the ash contains 10 to 50% Ni,so that it can be used as a „bio-ore“.
Such a plant mine can, according to field studies under commercial conditions,yield around 170 kg Ni per hectare and year. At the current (average Jan-May 2008) Ni price of around 18 € per kg raw nickel these are about 3000 € per hectare and year.
Phytomining pictures from R. Chaney
The location: a base-metal smelter, South Africa
The solution: phytoextraction usinga native nickel-accumulating species
The problem: Ni contamination over 5ha due to Ni salt storage and spillage
Phytoextraction in action
From: Presentation of Chris Anderson at CERM3 meeting
Interactions of plants with potentially toxic metals
Heavy metals in soil or water
root uptake exclusion mechanisms
damage to roots
root
± root to shoot translocation
stem epidermis
regulation by boundary cells?
stem xylem
stem phloem
leaf xylemleaf phloem
photosynthetic cells(e.g. mesophyll cells,stomatal guard cells)
epidermis cells(not present e.g. in Elodea)
vacuole
cell walls
cell walls vacuole
in the case of submerged
aquatic plants
metal-induced damage tophotosynthesis
(e.g. Mg-substitution)
cyto-plasm
vacuole cytoplasm
cell walls
oxidative stress and other damagecomplexation by strong
ligands (e.g.phytochelatins)
complexation by strong ligands (e.g.
phytochelatins)