Cadmium in plants – highly toxic but also
b
beneficial
fi i l
Elisa Andresen, Advanced Course on Bioinorganic Chemistry & Biophysics of Plants, summer semester 2012
Cadmium
www.webelements.com
Cadmium in the environment
• Rather rare element in Earth‘s crust (0.1
(0 1 ‐ 0.5
05
ppm)
• Some natural sites, associated with ZnS
• Anthropogenic contamination, e.g. ore
mining some fertilizers,
mining,
fertilizers car traffic,
traffic cigarette
smoke, industrial waste, NiCd‐batteries
www.wikipedia.com
Cd toxicity – prominent diseases
• Itai
Itai‐itai
itai disease (japanese ouch‐ouch
ouch ouch sickness)
• 1 of the 4 big Pollution Diseases in Japan
• Mass Cadmium poisoning in Japan, Cadmium
release into rivers by mining
• Severe pains in joints and spine,
spine softening of
the bones, kidney failure
• The mining companies were successfully sued
for the damage.
damage
Cadmium toxicity in plants
• Specific toxic effect often hard to measure
• E.g. growth a very unspecific effect.
– Less growth because Cd inhibits photosynthesis,
or respiration,
i ti
or uptake
t k off other
th nutrients,
t i t or …
Growth
1 week of 500 nM Cd leads to complete disruption of the plant
Growth
Before treatment start
After
f 3 weeks
k
After 1 week
After 2 weeks
After
f 4 weeks
k
C. demersum treated with 200 nM Cd for 4 weeks – Andresen et al. 2012, unpublished
Cadmium toxicity in plants –
1: Roots
First organ which gets affected
Reduced growth after Cd treatment
9 µM
45 µM
More layers
y of hypodermal
yp
periderm
p
More layers &
suberized cell
walls
ll (*) like
lik
after injury of
root surface
Lux et al., Annaly of Botany 107:285‐292, 2011
‐ Maize seedlings with proper roots placed between 2 agar blocks
‐ one of which contained Cd (50 or 100 µM), grown in phytochamber under
nature‐like conditions
 Roots bending towards the Cd‐containing agar  due to growth stop on the
Cd‐side & continued growth on control‐side
Lignification on Cd‐exposed
side
(*) and initiation of lateral
root primordium
(lrp)
Lux et al., Journal of Experimental Botany 62(1): 21‐37, 2011
Maize seedlings with proper roots placed between 2 agar blocks, one of which
contained Cd ((50 or 100 µ
µM))
Lignification at Cd‐exposed side
Gradual development of endodermal
suberin lamellae in untreated roots
In Cd‐exposed roots, suberin already 5mm
from apex (F), but not further away from
apex (E,D)
(E D)
 Suberin formation + lignification to
reduce unspecific permeability of root
membranes
Lux et al., Journal of Experimental Botany 62(1): 21‐37, 2011
Changes in the Root proteome after exposure to Cd
Roth et al., Journal of Experimental Botany 57(15):4003–4013, 2006
Cadmium toxicity in plants –
2: Photosynthesis
h
h
• Indirect measurement:
measurement Growth,
Growth O2 production
/ CO2 consumption
– Diminishing the Chl/pigment/protein content
• Di
Direct: Photosynthetic
Ph
h i paramters via
i
p y fluorescence measurement
Chlorophyll
How does Cd inhibit PS?
• Substitution of Mg2+ in Chl makes it unsuitable
for photosynthesis
•
•
unstable singlet excited state  black holes for excitons
shift of absorbance / fluorescence bands  energy transfer disturbed
•
different structure  proteins denature
•
when in reaction centre  charge separation prevented
Review: Küpper H, Küpper FC, Spiller M in Advances in Photosynthesis and Respiration, Kluwer Academic
Publishers, Dordrecht; pp. 67‐77, 2006
Measuring Chl fluorescence
Kautsky induction
 Photochemistry
 Heat dissipation
 Phosphorescence
 Fluorescence
uo esce ce
Andresen et al. 2012, unpublished
Pn (µm
mol CO2 m‐2 s‐1)
Measurement of net photosynthetic rate
www.adc.co.uk/Products/
T
Tomato
t plants
l t treated
t t d with
ith 5 ‐ 100 µM
M Cd for
f 7 days
d
showed
h
d reduced
d d Pn.
P
Haouari et al., African Journal of Plant Science 6(1):1‐7, 2012
Interference of Cd with PS II
PSII membrane fragments from spinach incubated with Cd (mM!! – Faller et al. 2004: µM do not
show inhibition in this system)
Not really physiological, only isolated PSII
mM concentrations  anyhow not whole plant, sufficient Cd to ensure reaction
0 M
0mM
50 M
50mM
100mM
Decrease in O2‐evolution  Cd
repleaces Ca in Mn‐cluster of water‐
splitting complex
Sigfridsson et al., Biochim & Biophys Acta 1659:19‐31, 2004
Shift of oxidation state of Cyt b559 in
presence of Cd  disturbance of protein
structure (loss of subunit) + replacement
of Ca in OEC
Cadmium toxicity in Plants –
3. ROS stress
Photosynthesis‐related
Photosynthesis
related ROS
Pospisil, Biochim & Biophys Acta 1817:218‐231, 2012
Pathogen‐related
Pathogen
related ROS – the oxidative burst
Wojtszek, Biochem J 322:681‐692, 1997
ROS and Cadmium
• Cadmium
Cadmi m redox
redo inert  No Fenton reaction!
Pinto, Journal of Phycology 39:1008‐1018, 2003
ROS and
d Cadmium
C d i
ROS production
Removal of ROS
• Cd interferes with
photosynthesis / respiration
 electrons transferred to
O2
• Cd replaces Zn in SOD (e.g.)
 less functional SOD
• In response antioxidant
y
enzymes
Ways to show Cd
Cd‐induced
induced ROS production
• Specific staining
H2O2 detection with DAB staining
pea plants grown with 50 µM Cd
Tabacco leaf discs exposed to 100 or 500µM
Cd for 3 hours, O2‐ staining with nitroblue
tetrazolium, DPI inhibits NADPH‐oxidase
dependant O2‐ formation
Romero‐Puertas et al., Plant, Cell, Envir.
27:1122‐1134, 2004
Iannone et al., Protoplasma 245:15‐27, 2010
Cadmium and ROS – Calcium limitation
Pea plants treated with
50 µM Cd
O2‐ (red) and NO (green)
NO‐synthase dependent
NO‐production depressed
by Cd, but effect prevented
by Ca.
Rodriguez‐Serrano et al.,
Plant Phys 150:229‐243, 2009
Ways to show Cd
Cd‐induced
induced ROS
ROS‐production
production
• D
Detection
i off Superoxide
S
id
formation with MCLA
(2‐metil‐6‐(4metoxipentil)‐3,7‐
dihydroimidazol 1,2‐apirazin‐3‐1
)
• Reaction of 1 molecule
O2‐ with 1 dye molecule
generates 1 photon
• Detect photon with
luminometre
hydrochlorhydrate
MCLA
Influence on antioxidant enzymes
Pea plants
0, 4, 40 µM Cd
Dixit et al., Journal of Experimental Botany
52(358):1101‐1109, 2001
Catalase: 2 H2O2 → 2 H2O + O2
Sandalio et al., Journal of Experimental Botany
52(364):2115‐2126, 2001
Influence on antioxidant enzymes
• Lower Cd concentrations and shorter
treatment duration tend to increase the
antioxidant system
• Longer
L
exposure and
d higher
hi h Cd
concentrations lead to decreased activityy or
content of the antioxidants
Cadmium toxicity in plants –
4. Genotoxicity
• Induction of DNA damage by
– direct interaction with the nucleotides
• modifications like base and sugar lesions, DNA strand
breaks, destruction of DNA‐protein
DNA protein crosslinks etc.
– inhibiting DNA repairing enzymes
– Induction of ROS, ROS lead to lipid peroxidation,
which causes membrane damage and production
of mutagenic aldehydes
Methods to detect Genotoxicity
• DNA Analyses
– Gelelectrophoresis and Comet Assay
– Random amplification of polymorphism DNA (RAPD)
• DNA / Chromosome Analyses
– Micronuclei formation
– Sister chromatid exchange
– Chromosomal aberrations
• Upregulation of DNA‐related / repairing enzymes
DNA disruption – Gelelectrophoresis and
Comet Assay
Lemna minor treated with µM concentrations of Cd
and Cu. Tail DNA (Comet ‐ length) increase due to
metal treatment.
Cvjetko et al., Arh Hig Rada Toksikol 61:287‐296, 2010
1: DNA from control plant, 2:‐6: DNA from plants
treated with Cd 10, 50, 75, 100, 1000 µM
Fojtova & Kovarik, Plant, Cell & Envir. 23:531‐537, 2000
From the CometAssay Manual, TrevigenR
Micronuclei & Mitotic index
• Vicia root meristem cells
• Mi
Micronuclei
l i formation
f
ti due
d
to malfunctioning cell
division
• Dose and time dependent
– Cd treatment increases MCN
• Mitotic index: ratio of cells
in metaphase stage to all
cells
– Cd treatment reduces MI
Souguir et al., Ecotoxicology 20:329‐336, 2011
Chromosomal aberrations
Vicia root meristem cells
a, b, e, f = 50 µM Cd
c d,
c,
d gg= 200 µM Cd
a & b = micronuclei
C = sticky chromosome
d = chromosome bridge
e = ““ + break
f = ““ +isolated
chromosome
g = laggered chromosome
in metaphase
Souguir et al., Ecotoxicology 20:329‐336, 2011
Sister chromatid exchange
‐Exchange
g of identical p
parts of both sister chromatids in the same
chromosome after / during DNA replication
‐As DNA sequence identical, exchange does not lead to genetic information
change
h
(≠ crossing
i over))
‐Happens in normal cells, but enhanced after treatment with toxic /
radioactive substances
FFrom: http://www.siteklabs.com/GenTox/
htt //
it kl b
/G T /
MammalianCellCytogenetics.html
Ünyayar et al., Turk J Biol 34:413‐422, 2010
Enhanced enzyme activity
Biphasic upregulation of dNTP‐providing
enzymes in A. thaliana treated with 25 µM
Cd.
Mediouni et al., Biosaline Agriculture and High
Salinity Tolerance, Birkhäuser Verlag, 2008
Telomerase activity in Tobacco BY‐2 cells
E 1:
Exp
1 4d off Cd treatment
t t
t (50 µM)
M) led
l d to
t
cell death
Exp 2: 3 days of Cd treatment (50 µM), 4
days of recovery in Cd‐free medium led to
increased Telomerase activity
Fojtova et al., Journal of Experimental Botany
53(378): 2151‐2158, 2002
Random amplification of polymorphism
DNA analyses
l
(RAPD)
(
)
• Cd interacts with DNA / induces mutation
• New
N / disappearing
di
i primer
i
bi di sites
binding
it
•  New / disappearing bands on gel
0
Shahrtash et al., J of Cell & Molecular Research 2(1):42‐48, 2010
360 µM
720µM
Cadmium toxicity in Plants –
5. Generall stress & stress prevention
Ph
Phytochelatins
h l i
• Cd binding to PC‐Synthase
induces synthesis of
Phytochelatins
• PC have high affinity to Cd
• Storage
g of PC‐Cd complexes
p
in vacuole
‐No
No PC
PC‐Cd
Cd complexes: extraction cannot ensure
that complexes were physiological
(Glu‐Cys)n‐Gly n=2‐11
http://www.chemie.uni‐oldenburg.de
/docs/forschungsberichte/fobe9596/3forsch.html
‐Higher
Higher Cd concentration  more different PCs
+ much higher amount of PC 2‐4
‐Threshold concentration 20 nM
Andresen et al., 2012, unpublished
No Cd
Cd‐CA
CA in Ceratophyllum?
• Treatment of C. demersum with 10 µM Cd (remember my
500 nM plant?)
– CA activity reduced with Cd, but enhanced with additional Zn
– Additional Zn removed Cd from the protein
Aravind & Prasad, J anal at spectrom 19:52‐57, 2004
Distribution of Cd in C. demersum
• µXRF reveals Cd,
Cd Zn & Mn distribution in Cd‐
Cd
treated leaves
From www.amptec‐ink.com
Andresen E, Küpper H (2013) Cadmium Toxicity in Plants. In: Cadmium: From Toxicity to Essentiality,
"Metal Ions in Life Sciences Vol. 11; in press
Beneficial effects of Cadmium in Plants
Beneficial effects of Cadmium
Ceratophyllum demersum forms longer lateral
shoots with 0.01 µg/ml = ~ 90nM Cadmium
Less Cd than we wanted..
Ornes & Sajwan, Water, Air, and Soil
Pollution 69: 291‐300, 1993.
Andresen et al. 2012, unpublished
Distribution of Cd in the oceans
• Micronutrient‐like
Micronutrient like
Abe et al., Global Environmental Changes in the Ocean and on Lands, TERRAPUB 189‐203, 2004
Cd‐Carbonic
Cd
Carbonic Anhydrase
‐Zn‐limited Thalassiosira weissflogii grow
better when Cd is added
‐A new CA protein for Cd is expressed
‐Cd‐CA larger than Zn‐CA
‐Cd‐CA
Cd CA can also
l bi
bind
d Zn
Z
‐Cd‐CA activity with Zn slightly, with Cd
much higher than Zn‐CA
Lane & Morel, PNAS 97(9):4627‐4631, 2000
Cd‐Carbonic
Cd
Carbonic Anhydrase
‐7
7 α‐helices,
h li
9 β‐sheets,
β h
bi di pocket
binding
k
‐ Active site: Cd bound to 2 Cys, 1 His, 1 H2O
‐Tetrahedral conformation
‐ (Acetate as substrate analogue)
Xu et al., Nature 452:56‐61, 2008
Take home messages
• Cadmium can affect a plant in various ways
– Induction of ROS  ROS react with everything in the cell
– Interfering with PS / respiration / metabolism
• Competing with other ions
• Replacing other ions in active centres of enzymes
• Cadmium can have a metabolic function under certain
circumstances
– Replacing other essential,
essential but missing ions
– Concentration dependant
– Hyperaccumulators
Thanks
Slides on the Küpper group homepage