The challenge of predicting metal transfer

through the soil-plant-animal continuum

Neal Menzies
Professor of Soil and Environmental Science
School of Agriculture and Food Sciences

The soil-plant-animal continuum

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 ?
Grazing animal

Vegetation cover

Contaminated soil

The soil-plant-animal continuum

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 ?
Grazing animal

Vegetation cover

Contaminated soil

The soil-plant-animal continuum

Need a relationship between metal extracted, and plant uptake
- Many extractants used

Metal in plant (mg/kg)

(strong acids, chelates, conc. salt solutions )

Metal extracted from soil (mg/kg)

Total metal content as a predictor

2500

(a)
2000

UQ study
Literature values

Plant tissue concentration (mg/kg)

1500

FAIL

1000

Animal toxicity threshold

Plant toxicity threshold

500

0
2500

(b)

2000

Monocots
Leafy vegetables
Dicots

1500

1000

500

0
0

250

500

750

1000

1250

Relationship between total metal content

and plant tissue concentration for
a. maize
b. all non-accumulator species

Total Zn (mg/kg)
Menzies et al 2007 Environ Pollut 145, 121-130

Common extractants as predictors

Plant tissue concentration (mg/kg)

FAIL
Animal toxicity threshold
Plant toxicity threshold

2500
Monocots
Leafy vegetables
Dicots

2000
1500
1000
500
0
0

250

500

750

1000

DTPA Extractable Zn (mg/kg)

1250

Relationship between metal extracted

and plant tissue concentration for
a. strong acid extraction
b. DTPA

Soils are too hard to deal with !

Lets make it simple
- plant metal uptake from solution
Grazing animal

Vegetation cover

Contaminated soil

Solution culture
Much simpler system
- but no agreement !

One experiment in detail (Pb)

Then an oversight of a lot of experiments
Kopittke et al 2010 J Exp Bot 61, 945-954

Pb How phyto-toxic is it?

The answer you get,
- depends on how you asked the question !
Literature values for Pb conc inducing toxicity span FOUR orders of magnitude

< 0.1 M (Kopittke et 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

Pb Phyto-toxicity in solution culture

Nutrient solution composition limits the max Pb exposure.
(b) Dilute Nutrient Solution

(a) Hoagland's Solution

Percentage of total Pb

100
Pb

75

Pb2+

Pb5(PO4)3Cl

2+

Pb5(PO4)3Cl

(precipitate)

(precipitate)

50
Experiments run at pH 5.5 and higher
have very little Pb in solution

25
0
3

5
Solution pH

Solution pH

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.
Kopittke et al Environ Pollut 153, 548-554

Pb Phyto-toxicity in solution culture

Nutrient solution composition limits the max Pb exposure.

Percentage of total Pb

100

(b) Dilute Nutrient Solution (PbCl2)

(a) Hoagland's Solution (PbCl2)

Pb5(PO4)3Cl

Pb5(PO4)3Cl

(precipitate)

(precipitate)

75
50
Pb2+

25
Pb2+
0
0.0

0.5

1.0
1.5
Initial Pb (M)

2.0

0.0

0.5

1.0
1.5
Initialsolution
Pb (M)
Even in dilute

2.0

Pbonconc
is Plimited
<0.5inM
Figure Predicted effects of the initial Pb conc
Pb and
species to
formed
(a) Hoaglands solution at pH 4.75 initially containing 1000 M P and 18 M Cl, and
(b) a dilute nutrient solution at pH 4.75 initially
containing precipitation
2 M P and 140kinetics
M Cl.
However,
are very slow

Pb toxicity in grass
Gross performance of Rhodes and signal grass.
Dilute nutrient solution culture experiments low P (2 M), low Cl (<1 M)
pH 4.75 maintained by NO3/ NH4 ratio and titration.
22L pots, 4 plants per pot = one replicate.
11 rates of Pb per species
Transplant seedlings at 7 days.
14 days growth in the test solutions

Rhodes grass the gold standard of tough grasses

Pb toxicity in grass

Relative fresh mass (%)

Gross performance of Rhodes and signal grass.

100
75

Shoots
Roots

Shoots
Roots

P < 0.001
R2 = 0.932

P < 0.001
2
R = 0.825

50
25
Rhodes grass

Signal grass
0
0

4
Pb

2+

(M)

12

Pb2+ (M)

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

12

Pb toxicity in grass
Gross performance of Rhodes and signal grass.
25

0.45

0.30

0.15
Rhodes grass
Signal grass
0.00

Rhodes grass
Signal grass

20

Root Pb (mg/g)

Shoot Pb (mg/g)

P < 0.001
2
R = 0.892

15
10
5

P < 0.001
2
R = 0.884

0
0

4
Pb

8
2+

(M)

12

4
Pb

8
2+

12

(M)

Figure The shoot and root tissue Pb concentrations of signal grass and
Rhodes grass after 14 d growth in dilute nutrient solutions

50% dry matter yield

Threshold for Pb in animal diets

Pb toxicity in grass
Rhodes grass root damage light microscopy
crystal violet stain.

0.5 M

0 M

1.1 M

Increasing damage to the root tip

- but the growth of root hairs continues unaffected

3.4 M

Pb toxicity in grass
Signal grass root damage light microscopy, crystal violet stain.

0 M

10 M

Much less damage to root growth than observed in Rhodes grass

Pb toxicity in grass
signal grass light microscopy, rhodizonate stain

0 M

0 M

10 M

10 M

10 M

Pb Phyto-toxicity in solution culture

Nutrient solution composition limits the max Pb exposure.

Percentage of total Pb

100

(b) Dilute Nutrient Solution (PbCl2)

(a) Hoagland's Solution (PbCl2)

Pb5(PO4)3Cl

Pb5(PO4)3Cl

(precipitate)

(precipitate)

75
50
Pb2+

25
Pb2+
0
0.0

0.5

1.0
1.5
Initial Pb (M)

2.0

0.0

0.5

1.0
1.5
Initialsolution
Pb (M)
Even in dilute

2.0

Pbonconc
is Plimited
<0.5inM
Figure Predicted effects of the initial Pb conc
Pb and
species to
formed
(a) Hoaglands solution at pH 4.75 initially containing 1000 M P and 18 M Cl, and
(b) a dilute nutrient solution at pH 4.75 initially
containing precipitation
2 M P and 140kinetics
M Cl.
However,
are very slow

Pb toxicity in grasses
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!

Tolerance physiology
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

Pb
This is the gene jockeys view!
If you look at up-regulation of genes
/ proteomics, this is what you see.

Pb-MT

Tolerance physiology
CW

Signal grass - TEM

1 mm behind root tip
Pb predominantly in cytoplasm
clearly present as a precipitate

C
V
2 m

C
CW
15 mm behind root tip
Pb predominantly in cell wall
precipitate particles larger, clearly crystalline
V
2 m
Kopittke et al 2008 Environ Sci Technol 42, 4595-4599

Reference materials
2000

Signal grass - TEM

Pb M1

Pb M1

Pb M1

2000
Counts

Counts

1500
1000

O
500

Energy dispersive X-ray (EDS) analysis

3000

Pb M1

O
1000

Cl

Cu

P
Cu

Pb M1

Pb M1

0
0

Pb5(PO4)3Cl Energy (keV)

Pb3(PO4)2 Energy (keV)

Reference materials
1000

chloropyromorphite (Pb5(PO4)3Cl)

750

Counts

lead phosphate (Pb3(PO4)2)

Pb

500

250

Plant samples

Cl

Cu
Ca

Lead is present as chloropyromorphite

0
0

Energy (keV)
400
C

Counts

300
Os
200
O
100

Pb

Cu

Cl

0
0

Energy (keV)

Tolerance physiology
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

Relative counts

1.00

Apoplastic-Pb
Pb5(PO4)3Cl

0.75

Pb3(PO4)2

ClK1

0.50

Pb M1

0.25
Pb M1

0.00
2.2

2.4

2.6

Energy (keV)

2.8

Tolerance physiology
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

Pb

Pb5(PO4)3Cl

The search for commonalities

We have spent a lot of time
looking at metal intoxicated roots

Kopittke et al Plant Soil 322, 303-315

The search for commonalities

We have spent a lot of time
looking at metal intoxicated roots
We became more and more interested in ruptures

The search for commonalities

We have spent a lot of time
looking at metal intoxicated roots
We became more and more interested in ruptures

Most metals cause ruptures

3.6 um La
But some do not

10 um Pb

The search for commonalities

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 cells
Cells 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 ?

The search for commonalities

Our experimental method
Short 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

One last shot at a common mechanism

THE DATASET

Ag

Al

Ba

Ca

Cd

Co

Cs

Cu

Ga

Gd

Hg

In

La

Li

Mg

Mn

Na

Ni

Pb

Ru

Sc

Sr

Tl

1mM Ca, 5um B

26 metals
6 rates
2 reps / 7 plants per rep
root length at 0 and 48h

Root elongation rate (mm/h)

Cowpea

Zn

Kopittke et al 2011 Environ Toxic Chem 30,

Concentration (M)
Most toxic Tl EC50b = 0.007 M
Least toxic K EC50b = 98,000 M

 con
Log EA50b (M)

Log EA500o (M)

Ionization energy

Consensus HL scale

Hydrated ionic radius

Log EA500o (M)

Standard electrode potential

Log EA500o (M)

Log EA500o (M)

Pauling electronegativity

Log EA500o (M)

Log EA500o (M)

Log EA500o (M)

One last shot at a common mechanism

Consensus HL scale

Covalent index

Expressing metal toxicity

Activity in solution
works the best
- but still not good!

Alva et al 1986 Commun Soil Sci Plant Anal 17, 1271-1280

Expressing metal toxicity

Activity at plasma membrane
- membrane has negative charge (attracts cations)

Relative root elongation

- we can alter membrane charge by altering solution ionic strength

1.0

(e)
Bulk solution activity

Activity at PM(f)

0.8

P < 0.001
R2 = 0.768

P < 0.001
R2 = 0.943

0.1 mM Ca
0.25 mM Ca
1 mM Ca
7.5 mM Ca
20 mM Ca

0.6
0.4
0.2
0.0
0.0

0.5

1.0

1.5

2.0

{Cu2+}Bulk (M)

2.5

3.0

40

80

120 160 200 240

o

{Cu2+}0 (M)

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

Expressing metal toxicity

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 (Al3+) reduce membrane charge
P < 0.001
R2 = 0.596

Relative root elongation

1.0

P < 0.001
R2 = 0.868

0.8
0.6

100

200

300

0.4
0.2
(f)

0.0 (e)
0.0

0.5

1.0
2+

1.5

2.0

{Cu }Bulk (M)

2.5

3.0

10

20

{Cu2+}0o (M)

30

40

 con
Log EA50b (M)

Log EA500o (M)

Ionization energy

Consensus HL scale

Hydrated ionic radius

Log EA500o (M)

Standard electrode potential

Log EA500o (M)

Log EA500o (M)

Pauling electronegativity

Log EA500o (M)

Log EA500o (M)

Log EA500o (M)

One last shot at a common mechanism

Consensus HL scale

Covalent index

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

One last shot at a common mechanism

General agreement for metals binding to strong ligands

(26 metals)

Metals binding strongly to soft ligands are a clear exception

A common mechanism some speculation

Metals prevent cell wall expansion causing ruptures
Metals binding to hard ligands (carboxyl)
(i) prevent acid loosening
(ii) prevent enzyme attach on pectins

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)

Supporting observations
Synchrotron
X-ray absorption (XAS)
- bonding arrangements
X-ray fluorescence (XRF)
- metal distribution

Do the metals behave as we predict ?

Bonding

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

Zn

Cu
Polygalacturonic acid
Phytic acid
Oxalic acid
Histidine
Cysteine
Citric acid
Aqueous
3h roots
24h roots

Bonding
X-ray absorption near edge structure (XANES)

extended X-ray absorption fine structure (EXAFS)

Cu

(c)

(a)

3 h Roots
Cysteine
Fitted

-3

k (k) ( )

Normalized x(E)

1.2

0.8

0.4
3 h Roots
Cysteine
Fitted

0.0

(d)

(b)

24 h Roots
Polygalacturonic acid
Fitted

-3

k (k) ( )

Normalized x(E)

1.2

0.8

0.4
24 h Roots
Polygalacturonic acid
Fitted

0.0
2

6
k (-1)

8960

8970

8980

8990
Energy (eV)

9000

9010

9020

Distribution

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

A common, nonspecific toxicity mechanism

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

Classification of metals according to bond

strength to hard and soft ligands.
Symbols indicate cowpea seedlings

showed ruptures
did not rupture