Heavy metal transport, accumulation and coordination in plants

– principles and methods

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)

Interactions of plants with potentially toxic metals

Heavy metals in soil or water

root uptake exclusionmechanisms

damage to roots

root

± root to shoottranslocation

stemepidermis

regulation byboundary cells?

stemxylem

stemphloem

leaf xylemleaf phloem

photosynthetic cells(e.g. mesophyll cells, stomatal guard cells)

epidermis cells

vacuole

cell walls

cell walls vacuole

in the case of submerged

aquatic plants

metal-induced damage to photosynthesis

(e.g. Mg-substitution)

cyto-plasm

vacuole cytoplasm

cell walls

oxidative stress and other damagecomplexation by strong

ligands

complexation by strongligands

Quantifying plant metal content – method of sample preparation

excise sample from plant, determine fresh weight

(freeze-) dry sample, then determine dry weight

add mixture of 85% concentrated nitric acid and 15% conc. perchloric acid

gradually (over 2.5h) heat until dry salt residue is achieved at 180 °C

or digest under pressure in microwave

re-disssolve in 5% hydrochloric acid, warm to 80°C, the dilute to 1.25% HCl

Metal content – methods of Measurement (I) Atomic Absorption Spectroscopy (AAS)

Advantages:- easy to use, - fast if only 1 element is needed

- affordable

Disadvantages:- insensitive for some elements (e.g. sulphur)

- slow if many elements are needed

Metal content – methods of Measurement (II)Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

Advantages:- Detection limits for most elements equal to or better than those obtained by Graphite Furnace –AAS (GFAAS)- Higher throughput than GFAAS- minimum of matrix interferences due to the high-temperature of the ICP source- Superior detection capability to ICP-AES with the same sample throughput- Ability to obtain isotopic information.

Disadvantages:- more complicated technique than AAS

- much more expensive than AAS- elements that prefer to form negative ions, such as Cl, I, F, etc. are very difficult to determine via ICP-MS because ions formed by the ICP discharge are typically positive ions.

argongas

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

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)

Methods of plant analysis using EDXASampling of single-cells saps with micropipettes

micropipette filled with silicon oil, connected to air-filled syringe for controlling pressure difference

turgor pressure of punctured cell fills pipette with 5-20 picolitres (10-12 l) of cell sap

Sample preparation:1) transfer to storage grid,addition of internal standard (e.g. RbF) and matrix (e.g. mannitol)

2) transfer to analysis grid, drying with isopentane

Analysis:1) recording of EDXA spectra in SEM2) data processing

typical dried sample20 µm

analysis grid

Methods of plant analysis using EDXAQuantification of elements in single-cells saps

1) net peak area is normalised by internal standard (an element not naturally present in the sample, e.g. Rb)

2) ratio obtained from 1) is quantified using calibration curve

1 10 100

1000

10000

100

Cadmium

coun

ts p

er 1

000

RbK

coun

ts

concentration / mM

Evaluation of the methodAdvantages:- potentially very accurate- enables measurement of

small concentrations

Disadvantages:- only few types of cells are accessible

to sampling with micropipettes- risk of preparation artefacts- no distinction between cytoplasm and

vacuole, measurement of cell walls impossible

- very difficult to obtain information about heterogeneity of element distribution inside the analysed tissue

Methods of plant analysis using EDXAFreeze-fracturing

Excise sample from plant, mount in/on stub or vice.

The EDXA spectrum of the vice must not interfere with that of the sample!

Freeze the sample in melting nitrogen slush, transfer to cooled (-170°C) preparation chamber

Fracture sample with fast-moving blade (to cut rather than break the cells)

Transfer to cooled (-150°C) sample stage in SEM, analyse

Produce conductive sample surface by evaporating carbon wire

Methods of plant analysis using EDXAAnalysis of bulk-frozen samples

Effect of shadingshading inside a sample leads to absorption of low-energy x-rays

Dot -map of O Kα line (0.6) Normal x-ray spectrumX-ray spectrum in shadow of trichome

Effect of acceleration voltagehigh acceleration voltage leads to deeper penetration into the sample!

sec. e-scatt. e-

inc. e-X-rays

fluorescence

ionisation volume

Methods of plant analysis using EDXAQualitative and semi-quantitative analysis of bulk-frozen samples

Line scansScan of the Zn K alpha line(0.6x half width) along the straight line. Amplitude represents the counts/s inside the selected spectral window.

Dot mapsScan of the Zn K alpha line(0.6x half width) over the whole image. Each dot represents one x-ray count inside the selected spectral window.

Methods of plant analysis using EDXAQuantitative analysis of bulk-frozen samples

Counts in spectra (A)can be normalised to either the background (B) or an internal standard. The oxygen Kα line has proven to be a reliable internal standard in bulk-frozen samples, in particular in aqueous compartments like vacuoles (C).

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 elements in leaves:Cells with differing physiology in the mesophyll of

metal-stressed hyperaccumulators

0 2 4 6 8 100

5

10

15

20

25

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

Mg

/ mM

Cd / mM

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

Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP (2001)

J Exp Bot 52 (365), 2291-2300

0.0 0.5 1.0 1.5 2.00

2

4

6

8

10

12

14

Mg

/ mM

Ni / mM

Arabidopsis halleri Thlaspi goesingense

Methods of plant analysis using EDXAAnalysis of bulk-frozen samples

Evaluation of the freeze-fracturing methodAdvantages:- All types of cells and tissues can be analysed - In situ-analysis with very little risk of preparation artefacts- Easy analysis of the heterogeneity of element distribution, by use of dot-maps even in

an imaging way

Disadvantages:- Limited sensitivity (min. 1mM) and accuracy (shading)- Elements in dead tissues with low water content cannot be reliably quantified

How Synchrotron radiation is generated

DESY

Bending Magnet

Wiggler

Undulator

Free Electron Laser

Preparation of plant material for XAS (EXAFS and XANES)

Excise sample from plant, mount in/on stub or vice.The EXAFS spectrum of the vice must not interfere with that of the sample!

Freeze the sample in melting nitrogen slush

grind sample in mortar cooled by dry ice

fill the still frozen-hydrated powder into an EXAFS cuvette, seal with Kapton tape

Transfer to cooled (20 K) sample holder of beamline, analyse

X-ray absorption (I)

X-ray absorption (II)

XAS techniques

What can we learn from XAS?

Example of what can we learn from XANES

Analysis of metal ligands byExtended X-ray Absorption

Fine Structure (EXAFS)

Principle of Extended X-ray Absorption Fine Structure (EXAFS)

Effects of single vs. multiple scattering contributions in EXAFS

26700 26800 26900 27000 271000.00.20.40.60.81.01.21.4

2 4 6 8 10 12

-6-4-20246

0 1 2 3 4 5 60

51015

202530

26750 268000.8

1.0

1.2 XAS

Nor

mal

ised

X-r

ay fl

uore

scen

ce

Excitation energy [eV]

k / Å-1

EXAFSEXA

FS [C

hi*k

3 ]

oxygen (aqueous) oxygen (citrate) histidine sulphur (glutathione)

EXAFS Fourier transform

Distance [Å]

Tran

sfor

m A

mpl

itude

26700 26800 26900 27000 271000.00.20.40.60.81.01.21.4

2 4 6 8 10 12

-4

-2

0

2

4

6

0 1 2 3 4 5 60

4

8

12

16

20

24

26750 26800

1.0

1.2

measured data fit with datasets of model compounds

XAS

Nor

mal

ised

X-r

ay fl

uore

scen

ce

Excitation energy [eV]

measured data fit with theoretical model

k / Å-1

EXAFSEXAF

S [C

hi*k

3 ]

measured data fit with theoretical model fit with datasets of

model compounds

EXAFS Fourier transform

Distance [Å]

Tran

sfor

m A

mpl

itude

Cd-ligands: model compounds Cd in hyperaccumulator leaves

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

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 ( Barbara’s lecture)

Leitenmaier B, Küpper H, (2007) unpublished

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

stor

age c

ells

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

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

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

UV/VIS absorbance spectroscopySpectra of Elodea canadensis extracts

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

In vivo UV/VIS absorbance spectroscopy

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

•different structure --> proteins denature

• do not readily perform charge separation when in reaction centre

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

S0

S2

S1T1

h·ν

h·ν

intersystem crossing

absorption

absorptionfluorescence

intersystem crossingintersystem crossing

phosphorescence

intersystem crossing

photochemistry

In vivo chlorophyll fluorescence

Fp

Static fluorescence microscopy of metal-stressed Elodea

red (650-700nm) chlorophyll fluorescence

transmittant light observation

1 µM Cu2+ control 100 µM Zn2+

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

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

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

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 Phytologist 175, 655-674

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

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

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

gen

prod

uctio

n/ %

of M

g-C

hl a

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

0

20

40

60

80

100

120

140

lifet

ime

of C

hl tr

iple

t exc

ited

stat

e/ %

of M

g-C

hl a

0

20

40

60

80

100

120

140

Chl a derivatives Chl b derivatives

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

lifet

ime

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

All slides of my lectures are available in the Internet:

http://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_education.html