Heavy Metals and Plants - a complicated relationship

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


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.


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


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!


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


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


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


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


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)

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Heavy Metals and Plants - a complicated relationship