The challenge of predicting metal transfer through the soil-plant-animal continuum
Transcript of 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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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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The soil-plant-animal continuum
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 !
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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)
Nutrient solution composition limits the max Pb exposure.
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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Gross performance of Rhodes and signal grass.
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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Gross performance of Rhodes and signal grass.
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
Pb toxicity in grass
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
Pb toxicity in grass
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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
Pb toxicity in grass
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signal grass light microscopy, rhodizonate stain
0 M 0 M
10 M 10 M 10 M
Pb toxicity in grass
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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)
Nutrient solution composition limits the max Pb exposure.
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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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
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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
Tolerance physiology
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
Tolerance physiology
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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
Tolerance physiology
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The search for commonalities
We have spent a lot of time
looking at metal intoxicated roots
Kopittke et al Plant Soil 322, 303-315
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The search for commonalities
We have spent a lot of time
looking at metal intoxicated roots
We became more and more interested in ruptures
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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
But some do not
10 um Pb
3.6 um La
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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 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 ?
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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
The search for commonalities
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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,
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One last shot at a common mechanism
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)
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Expressing metal toxicity
Activity in solution
works the best
- but still not good! Alva et al1986 Commun Soil Sci Plant Anal 17, 1271-1280
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Expressing metal toxicity
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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Expressing metal toxicity
{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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One last shot at a common mechanism
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)
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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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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
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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
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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)
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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