the challenge of predicting metal transfer through the soil-plant-animal continuum

7/24/2019 The challenge of predicting metal transfer through the soil-plant-animal continuum

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Neal Menzies

Professor of Soil and Environmental Science

School of Agriculture and Food Sciences

The challenge of predicting metal transfer

through the soil-plant-animal continuum


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The soil-plant-animal continuum

Contaminated soil

Vegetation cover

Grazing animal

Wish to predict on the basis of a simple measure

-How much metal will get into the plant ?

-How much will get into the animal ?


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Contaminated soil

Vegetation cover

Grazing animal

Wish to predict on the basis of a simple measure

-How much metal will get into the plant ?

-How much will get into the animal ?


4/43


Need a relationship between metal extracted, and plant uptake

- Many extractants used

(strong acids, chelates, conc. salt solutions )

Metal extracted from soil (mg/kg)

Metalinplant(mg/kg)


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P

lanttissueconcentration(mg/kg)

0

500

1000

1500

2000

2500

UQ study

Literature values

Total Zn (mg/kg)

0 250 500 750 1000 1250

0

500

1000

1500

2000

2500

Monocots

Leafy vegetables

Dicots

(a)

(b)

Animal toxicity threshold

Plant toxicity threshold

Relationship between total metal content

and plant tissue concentration for

a. maize

b. all non-accumulator species

FAIL

Total metal content as a predictor

Menzies et al 2007 Environ Pollut 145, 121-130


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Common extractants as predictors

DTPA Extractable Zn (mg/kg)

0 250 500 750 1000 1250

Pl

anttissueconcentration(mg/kg)

0

500

1000

1500

2000

2500

Monocots

Leafy vegetables

Dicots

Animal toxicity threshold

Plant toxicity threshold

Relationship between metal extracted

and plant tissue concentration for

a. strong acid extraction

b. DTPA

FAIL


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Soils are too hard to deal with !

Lets make it simple- plant metal uptake from solution

Contaminated soil

Vegetation cover

Grazing animal


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One experiment in detail (Pb)

Then an oversight of a lot of experiments

Kopittke et al 2010 J Exp Bot 61, 945-954

Solution culture

Much simpler system

- but no agreement !


9/43

Literature values for Pb conc inducing toxicity span FOURorders of magnitude

< 0.1 M (Kopittkeet al. Environ. Poll

. 2007) > 1000 M (Yang et al. J. Environ. Sci. 2001)

Some of this difference may be difference in species tolerance

But much relates to poor experimental practice.- the most common error being nutrient solutions containing high P

The answer you get,- depends on how you asked the question !

Pb How phyto-toxic is it?


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Nutrient solution composition limits the max Pb exposure.

Figure. Predicted effects of solution pH on distribution of the total Pb for (a) a Hoaglands

solution containing 1 M Pb, 2000 M P, and 18 M Cl, and (b) a dilute nutrient solution

containing 1 M Pb, 2 M P, and 200 M Cl.

Solution pH

3 4 5 6 7

PercentageoftotalPb

0

25

50

75

100

Pb2+

(a) Hoagland's Solution

Solution pH

3 4 5 6 7

Pb2+

(b) Dilute Nutrient Solution

Pb5(PO

4)3Cl

(precipitate)

Pb5(PO

4)3Cl

(precipitate)

Experiments run at pH 5.5 and higher

have very little Pb in solution

Pb Phyto-toxicity in solution culture

Kopittke et al Environ Pollut 153, 548-554


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Initial Pb (M)

0.0 0.5 1.0 1.5 2.0

Percentageo

ftotalPb

0

25

50

75

100

Pb5(PO

4)3Cl

(precipitate)

Pb2+

(a) Hoagland's Solution (PbCl2)

Initial Pb (M)

0.0 0.5 1.0 1.5 2.0

Pb2+

(b) Dilute Nutr ient Solution (PbCl2)

Pb5(PO

4)3Cl

(precipitate)


Figure Predicted effects of the initial Pb conc on Pb and P species formed in

(a) Hoaglands solution at pH 4.75initially containing 1000 M P and 18 M Cl, and

(b) a dilute nutrient solution at pH 4.75 initially containing 2 M P and 140 M Cl.

Even in dilute solution

Pb conc is limited to


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Gross performance of Rhodes and signal grass.

Dilute nutrient solution culture experiments low P (2 M), low Cl (


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Signal grass

Pb2+

(M)

0 4 8 12

Relativefreshma

ss(%)

0

25

50

75

100 Shoots

Roots

Rhodes grass

Pb2+

(M)

0 4 8 12

Shoots

Roots

P < 0.001R

2= 0.932

P < 0.001

R2= 0.825

Figure The relative fresh mass of the roots and shoots of signal grass (left)

and Rhodes grass (right) after 14 d growth in dilute nutrient solutions

Pb toxicity in grass


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FigureThe shoot and root tissue Pb concentrations of signal grass and

Rhodes grass after 14 d growth in dilute nutrient solutions

Pb2+(M)

0 4 8 12

ShootPb

(mg/g)

0.00

0.15

0.30

0.45

Rhodes grass

Signal grass

Pb2+(M)

0 4 8 12

RootPb(mg/g)

0

5

10

15

20

25

Rhodes grass

Signal grass

P < 0.001

R2= 0.892

P < 0.001

R2= 0.884

Threshold for Pb in animal diets


50% dry matter yield


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Rhodes grass root damage light microscopycrystal violet stain.

0 M

0.5 M

1.1 M

3.4 M

Increasing damage to the root tip

- but the growth of root hairs continues unaffected



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Signal grass root damage light microscopy, crystal violet stain.

Much less damage to root growth than observed in Rhodes grass

0 M 10 M



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signal grass light microscopy, rhodizonate stain

0 M 0 M

10 M 10 M 10 M



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Initial Pb (M)

0.0 0.5 1.0 1.5 2.0

Percentageofto

talPb

0

25

50

75

100

Pb5(PO

4)3Cl

(precipitate)

Pb2+

(a) Hoagland's Solution (PbCl2)

Initial Pb (M)

0.0 0.5 1.0 1.5 2.0

Pb2+

(b) Dilute Nutr ient Solution (PbCl2)

Pb5(PO

4)3Cl

(precipitate)


Figure Predicted effects of the initial Pb conc on Pb and P species formed in

(a) Hoaglands solution at pH 4.75initially containing 1000 M P and 18 M Cl, and

(b) a dilute nutrient solution at pH 4.75 initially containing 2 M P and 140 M Cl.

Even in dilute solution

Pb conc is limited to


19/43

signal grass light microscopy, rhodizonate stain

0 M 0 M

10 M 10 M 10 M

This is not a tolerance strategy its an artefact!

Pb toxicity in grasses


20/43

Pb Pb

Standard model of plant response

Pb enters cell cytoplasm

Cell responds by producing metallothionein (MT)

MT complexes Pb, detoxifying it.

Pb stays in solution

MT

-MT

Tolerance physiology

This is the gene jockeys view!

If you look at up-regulation of genes

/ proteomics, this is what you see.


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V

CW

C

V

2 m

2 m

CW

V

V

C

Signal grass - TEM

1 mm behind root tip

Pb predominantly in cytoplasm

clearly present as a precipitate

15 mm behind root tip

Pb predominantly in cell wall precipitate particles larger, clearly crystalline


Kopittke et al 2008 Environ Sci Technol 42, 4595-4599


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Signal grass - TEM

Energy (keV)

0 1 2 3 4 5

Counts

0

250

500

750

1000

O

Cu

P

Pb

Cl

Ca

Energy (keV)

0 1 2 3 4 5

Counts

0

100

200

300

400

OCu

PbCl

Os

C

Energy (keV)

0 1 2 3 4 5

Counts

0

500

1000

1500

2000

O

Cu

PCl

Pb 1M

Pb 1M

Pb 1M

Energy (keV)

0 1 2 3 4 5

Counts

0

1000

2000

3000Pb 1M

Pb 1M

O

Cu

P

Pb 1M

Pb5(PO4)3Cl Pb3(PO4)2

Reference materials

O

O

Energy dispersive X-ray (EDS) analysis

Reference materials

chloropyromorphite (Pb5(PO4)3Cl)

lead phosphate (Pb3(PO4)2)

Plant samples

Lead is present as chloropyromorphite


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Energy (keV)

2.2 2.4 2.6 2.8

Relativecoun

ts

0.00

0.25

0.50

0.75

1.00Apoplastic-Pb

Pb5(PO

4)3Cl

Pb3

(PO4

)2

Pb 1M

Pb 1M1KCl

Chloropyromorphite

Confirmation of mineral form

correct morphology (hex needles)

repeated EDS analysis (10 reps) same result from EELS on cryo. samples

(electron energy loss)

Why chloropyromorphite ?Lowest solubility of Pb forms



24/43

Pb Pb5(PO4)3Cl

Proposed model of plant response in signal grass

Pb enters cell cytoplasm

Pb is precipitated as chloropyromorphite

Precipitation reduces soluble Pb in cytoplasm

Solid is moved to cell walls

Golgi apparatus may have a role

in moving the solid to the cell wall



25/43

The search for commonalities

We have spent a lot of time

looking at metal intoxicated roots

Kopittke et al Plant Soil 322, 303-315


26/43




We became more and more interested in ruptures


27/43




We became more and more interested in ruptures

Most metals cause ruptures

But some do not

10 um Pb

3.6 um La


28/43


Our working hypothesis for toxicity of metals

Metals bind to the walls of cells in the rhizodermis and outer cortex.

This increases cell wall rigidity in the zone of elongation

Ruptures form due to the presence of rigid (slowly expanding) outer cellsCells of the stele and inner cortex expand at a faster rate.

Ruptures form as the inner expansion breaks the rigid outer layer

How do we expand this to accommodate non-rupturing metals ?


29/43

Our experimental methodShort term experiments

Cowpea (most commonly)

Root elongation as plant growth measure

Dilute solution culture

Complete nutrient suite (usually)

- 1mM Ca and 5 uM B as minimum

Measurement of actual conc. present

Calculation of activity in solution

Calculation of activity at plasma membrane



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Ag Al

Co Cs

Ba Ca Cd

Cu Ga Gd

Li Mg Mn

HgH

Na

In K La

Ni

Zn

Pb Ru Sc Sr Tl

One last shot at a common mechanism

THE DATASET

Cowpea

1mM Ca, 5um B

26 metals

6 rates

2 reps / 7 plants per rep

root length at 0 and 48h

Rootelongationra

te(mm/h)

Concentration (M)

Most toxic Tl EC50b= 0.007 M

Least toxic K EC50b= 98,000 MKopittke et al 2011 Environ Toxic Chem 30,


31/43


Pauling electronegativity Standard electrode potential Hydrated ionic radius

Ionization energy Covalent indexcon

Consensus HL scale Consensus HL scale

LogEA500

o(

M)

LogEA500

o(

M)

LogEA500

o(

M)

LogEA500o

(M)

LogEA500o

(M)

LogEA500o

(M)

LogEA500

o(

M)

LogEA50b

(M)


32/43

Expressing metal toxicity

Activity in solution

works the best

- but still not good! Alva et al1986 Commun Soil Sci Plant Anal 17, 1271-1280


33/43


Activity at plasma membrane

- membrane has negative charge (attracts cations)

- we can alter membrane charge by altering solution ionic strength

{Cu2+

}0

o(M)

0 40 80 120 160 200 240

0.1 mM Ca0.25 mM Ca

1 mM Ca

7.5 mM Ca

20 mM Ca

{Cu2+

}Bulk

(M)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Relativerootelongation

0.0

0.2

0.4

0.6

0.8

1.0P < 0.001

R2= 0.768

P < 0.001

R2= 0.943

(f)(e)Bulk solution activity Activity at PM

Kopittke et al 2011 Environ Sci Technol 45, 4966-4973


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{Cu2+

}0

o(M)

0 10 20 30 40

{Cu2+

}Bulk

(M)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Relativerootelongation

0.0

0.2

0.4

0.6

0.8

1.0 P < 0.001R

2= 0.596

0 100 200 300

P < 0.001

R2= 0.868

(f)(e)

Activity at plasma membrane

- membrane has negative charge (attracts cations)

We can alter membrane charge by

-changing solution ionic strength

- altering solution pH

- strongly adsorbing cations (Al

3+

) reduce membrane charge


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Pauling electronegativity Standard electrode potential Hydrated ionic radius

Ionization energy Covalent indexcon

Consensus HL scale Consensus HL scale

LogEA500

o(

M)

LogEA500

o(

M)

LogEA500

o(

M)

LogEA500

o(

M)

LogEA500

o(

M)

LogEA500

o(

M)

LogEA500

o(

M)

LogEA50b

(M)


36/43

Metal bonding to cell walls

A common, nonspecific mechanism of toxicity ?

This should be predictable.

Rupturing for metals which form strong bonds with cell wall components

Classification of metals according to bond

strength to hard and soft ligands.

Symbols indicate cowpea seedlings

showed ruptures

did not rupture

Hard ligands - carboxyl, hydroxyl, phosphoryl, sulfate, and amine groups

Soft ligands - sulfhydryl groups, olefins, or aromatic groups


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General agreement for metals binding to strong ligands (26 metals)

Metals binding strongly to soft ligands are a clear exception


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A common mechanism some speculation

Metals prevent cell wall expansion causing ruptures

Metals binding soft ligands (eg. Ag)

(i) interference with basipetal auxin flow

- inhibits the transport of IAA-across the plasmalemma

- interferes with H+

-ATPase

(ii) strong binding to proteins with soft-ligand moieties

(e.g., endoglucanases, expansins)

Metals binding to hard ligands (carboxyl)

(i) prevent acid loosening

(ii) prevent enzyme attach on pectins


39/43

Supporting observations

SynchrotronX-ray absorption (XAS)

- bonding arrangements

X-ray fluorescence (XRF)

- metal distribution

Do the metals behave as we predict ?

B di K edge extended X ray absorption fine structure (EXAFS)


40/43

Zn CuBonding

Polygalacturonic acid

Phytic acid

Oxalic acid

Histidine

Cysteine

Citric acid

Aqueous

3h roots

24h roots

K-edge extended X-ray absorption fine structure (EXAFS)


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k (-1)

2 4 6 8

k3

(k)(-3)

24 h RootsPolygalacturonic acidFitted

k3

(k)(-3)

3 h RootsCysteineFitted

Energy (eV)

8960 8970 8980 8990 9000 9010 9020

Normalized

x(E)

0.0

0.4

0.8

1.2

24 h RootsPolygalacturonic acidFitted

Normalizedx(E)

0.0

0.4

0.8

1.2

3 h Roots

CysteineFitted

(a)

(b) (d)

(c)Cuextended X-ray absorption fine structure (EXAFS) X-ray absorption near edge structure (XANES)

Bonding


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Distribution

Wang et al 2013 Sci Total Environ 463-464:131-139.


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A common, nonspecific toxicity mechanism

Classification of metals according to bond

strength to hard and soft ligands.

Symbols indicate cowpea seedlings

showed ruptures

did not rupture

Classification of metals according to affinity to ligandsRelationships based on activity at the plasma membrane

the challenge of predicting metal transfer through the soil-plant-animal continuum

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