Cadmium in plants – highly toxic but also b fi i lbeneficial

Elisa Andresen, Advanced Course on Bioinorganic Chemistry  & Biophysics of Plants, summer semester 2012

CadmiumCadmium

www.webelements.com

Cadmium in the environmentCadmium in the environment

• Rather rare element in Earth‘s crust (0 1 0 5• Rather rare element in Earth‘s crust (0.1 ‐ 0.5 ppm)

• Some natural sites, associated with ZnS• Anthropogenic contamination, e.g. oremining some fertilizers car traffic cigarettemining, some fertilizers, car traffic, cigarettesmoke, industrial waste, NiCd‐batteries

www.wikipedia.com

Cd toxicity – prominent diseasesCd toxicity prominent diseases

• Itai itai disease (japanese ouch ouch sickness)• Itai‐itai disease (japanese ouch‐ouch sickness)• 1 of the 4 big Pollution Diseases in Japan1 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 softening of• Severe pains in joints and spine, softening ofthe bones, kidney failure

• The mining companies were successfully suedfor the damagefor the damage.

Cadmium toxicity in plantsCadmium toxicity in plants

• Specific toxic effect often hard to measure• Specific toxic effect often hard to measure• E.g. growth a very unspecific effect.E.g. growth a very unspecific effect. 

– Less growth because Cd inhibits photosynthesis, i ti t k f th t i tor respiration, or uptake of other nutrients, or …

GrowthGrowth

1 week of 500 nM Cd leads to complete disruption of the plant

GrowthGrowthBefore treatment start After 1 week After 2 weeks

f k f kAfter 3 weeks After 4 weeks

C. demersum treated with 200 nM Cd for 4 weeks – Andresen et al. 2012, unpublished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 treatmentMore layers of hypodermal peridermy yp p

9 µM 45 µM

More layers & suberized cell

ll (*) likwalls (*)  likeafter injury ofroot 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 undernature‐like conditions

Roots bending towards the Cd‐containing agar due to growth stop on theCd‐side & continued growth on control‐side

Lignification on Cd‐exposedside(*) and initiation of lateral root primordium(lrp)(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 whichcontained Cd (50 or 100 µM)( µ )

Gradual development of endodermal suberin lamellae in untreated roots

Lignification at Cd‐exposed side

Suberin formation + lignification toreduce unspecific permeability of rootmembranes

In Cd‐exposed roots, suberin already 5mm from apex (F), but not further away from

(E D)

Lux et al., Journal of Experimental Botany 62(1): 21‐37, 2011

membranesapex (E,D) 

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 –h h2: Photosynthesis

• Indirect measurement Growth O production• Indirect measurement: Growth, O2 production/ CO2 consumption2

– Diminishing the Chl/pigment/protein content

Di Ph h i i• Direct: Photosynthetic paramters via Chlorophyll fluorescence measurementp y

How does Cd inhibit PS?How does Cd inhibit PS? 

• Substitution of Mg2+ in Chl makes it unsuitable• Substitution of Mg2+ in Chl makes it unsuitablefor 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 fluorescenceMeasuring Chl fluorescence

Kautsky induction

Photochemistry Heat dissipation Phosphorescence Fluorescenceuo esce ce

Andresen et al. 2012, unpublished

Measurement of net photosynthetic rateMeasurement of net photosynthetic ratem

‐2s‐1)

molCO

2Pn

(µm

www.adc.co.uk/Products/

T t l t t t d ith 5 100 M Cd f 7 d h d d d PTomato plants treated with 5 ‐ 100 µM Cd for 7 days showed reduced Pn.

Haouari et al., African Journal of Plant Science 6(1):1‐7, 2012

Interference of Cd with PS IIInterference of Cd with PS IIPSII 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 PSIImM concentrations anyhow not whole plant, sufficient Cd to ensure reaction

0 M0mM

50 M50mM

100mM

Decrease in O2‐evolution  Cdrepleaces Ca in Mn‐cluster of water‐splitting complex

Shift of oxidation state of Cyt b559 in presence of Cd disturbance of proteinstructure (loss of subunit) + replacement

Sigfridsson et al., Biochim & Biophys Acta 1659:19‐31, 2004

splitting complex structure (loss of subunit) + replacementof Ca in OEC

Cadmium toxicity in Plants –3. ROS stress

Photosynthesis‐related ROSPhotosynthesis related ROS

Pospisil, Biochim & Biophys Acta 1817:218‐231, 2012

Pathogen‐related ROS – the oxidative burstPathogen related ROS  the oxidative burst

Wojtszek, Biochem J 322:681‐692, 1997

ROS and CadmiumROS and Cadmium

• Cadmi m redo inert No Fenton reaction!• Cadmium redox inert  No Fenton reaction!

Pinto, Journal of Phycology 39:1008‐1018, 2003

ROS d C d iROS and Cadmium

ROS production Removal of ROS 

• Cd interferes with • Cd replaces Zn in SOD (e.g.) photosynthesis / respiration electrons transferred to

less functional SOD

O2

• In response antioxidantenzymesy

Ways to show Cd‐induced ROS productionWays to show Cd induced ROS production

• Specific staining

H2O2 detection with DAB stainingTabacco leaf discs exposed to 100 or 500µM Cd for 3 hours, O2

‐ staining with nitrobluepea plants grown with 50 µM Cd

2tetrazolium, DPI  inhibits NADPH‐oxidasedependant 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 limitationCadmium and ROS  Calcium limitation

Pea plants treated with50 µM Cd50 µM Cd

O2‐ (red) and NO (green)

NO‐synthase dependentNO‐production depressedby Cd, but effect preventedby Ca.

Rodriguez‐Serrano et al., Plant Phys 150:229‐243, 2009

Ways to show Cd‐induced ROS‐productionWays to show Cd induced ROS production

D i f S id• Detection of Superoxide formation with MCLA  (2‐metil‐6‐(4metoxipentil)‐3,7‐dihydroimidazol 1,2‐apirazin‐3‐1 

)hydrochlorhydrate)• Reaction of 1 molecule

MCLA

O2‐ with 1 dye molecule

generates 1 photongenerates 1 photon• Detect photon withluminometre

Influence on antioxidant enzymesInfluence on antioxidant enzymes

Pea plants0, 4, 40 µM Cd

Catalase: 2 H2O2 → 2 H2O + O2

Sandalio et al., Journal of Experimental Botany52(364):2115‐2126, 2001 

Dixit et al.,  Journal of Experimental Botany52(358):1101‐1109, 2001

Influence on antioxidant enzymesInfluence on antioxidant enzymes

• Lower Cd concentrations and shorter• Lower Cd concentrations and shortertreatment duration tend to increase the antioxidant system L d hi h Cd• Longer exposure and higher Cdconcentrations lead to decreased activity or ycontent of the antioxidants

Cadmium toxicity in plants –4. Genotoxicity

• Induction of DNA damage by• Induction of DNA damage by– direct interaction with the nucleotides

• modifications like base and sugar lesions, DNA strand breaks, destruction of DNA‐protein crosslinks etc.breaks, destruction of 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 GenotoxicityMethods to detect Genotoxicity

• DNA Analyses• DNA Analyses– Gelelectrophoresis and Comet Assay– Random amplification of polymorphism DNA (RAPD)

• DNA / Chromosome Analyses• DNA / Chromosome Analyses– Micronuclei formation– Sister chromatid exchange– Chromosomal aberrationsChromosomal aberrations

• Upregulation of DNA‐related / repairing enzymes

DNA disruption – Gelelectrophoresis andComet Assay

Lemna minor treated with µM concentrations of Cdand Cu. Tail DNA (Comet ‐ length) increase due tometal treatment.

Cvjetko et al., Arh Hig Rada Toksikol 61:287‐296, 2010

1: DNA from control plant, 2:‐6: DNA from plantstreated with Cd 10, 50, 75, 100, 1000 µM

Fojtova & Kovarik, Plant, Cell & Envir. 23:531‐537, 2000

From the CometAssay Manual, TrevigenR

Micronuclei & Mitotic indexMicronuclei & Mitotic index

• Vicia root meristem cells• Vicia root meristem cells

Mi l i f ti d• Micronuclei formation due to malfunctioning celldivisiondivision

• Dose and time dependent– Cd treatment increases MCN– Cd treatment increases MCN

• Mitotic index: ratio of cells• Mitotic index: ratio of cellsin metaphase stage to all cells– Cd treatment reduces MI

Souguir et al., Ecotoxicology 20:329‐336, 2011

Chromosomal aberrationsChromosomal aberrations

Vicia root meristem cellsa, b, e, f = 50 µM Cdc d g 200 µM Cdc, d, g= 200 µM Cd

a & b = micronucleiC = sticky chromosomed = chromosome bridgee = ““ + breakf = ““ +isolatedchromosomeg = laggered chromosomeg   laggered chromosomein metaphase

Souguir et al., Ecotoxicology 20:329‐336, 2011

Sister chromatid exchangeSister chromatid exchange‐Exchange of identical parts of both sister chromatids in the same g pchromosome after / during DNA replication‐As DNA sequence identical, exchange does not lead to genetic informationh (≠ i )change (≠ crossing over)‐Happens in normal cells, but enhanced after treatment with toxic / radioactive substances

F htt // it kl b /G T /From: http://www.siteklabs.com/GenTox/MammalianCellCytogenetics.html

Ünyayar et al., Turk J Biol 34:413‐422, 2010

Enhanced enzyme activityEnhanced enzyme activity

Telomerase activity in Tobacco BY‐2 cellsE 1 4d f Cd t t t (50 M) l d t

Biphasic upregulation of dNTP‐providingenzymes in A. thaliana treated with 25 µM 

Exp 1:  4d of Cd treatment (50 µM) led tocell deathExp 2: 3 days of Cd treatment (50 µM), 4 

Cd. days of recovery in Cd‐free medium led toincreased Telomerase activity

Mediouni et al., Biosaline Agriculture and High Salinity Tolerance, Birkhäuser Verlag, 2008

Fojtova et al., Journal of Experimental Botany 53(378): 2151‐2158, 2002

Random amplification of polymorphism l ( )DNA analyses (RAPD)

• Cd interacts with DNA / induces mutationN / di i i bi di it• New / disappearing primer binding sites

• New / disappearing bands on gel0 360 µM 720µM

New / disappearing bands on gel

Shahrtash et al.,  J of Cell & Molecular Research 2(1):42‐48, 2010 

Cadmium toxicity in Plants –l5. General stress & stress prevention

Ph h l iPhytochelatins• Cd binding to PC‐Synthase

induces synthesis ofPhytochelatins

• PC have high affinity to Cd• Storage of PC‐Cd complexesg p

in vacuole‐No PC‐Cd complexes: extraction cannot ensureNo PC Cd complexes: extraction cannot ensurethat complexes were physiological

Higher Cd concentrationmore different PCs(Glu‐Cys)n‐Gly n=2‐11

‐Higher Cd concentrationmore different PCs  + much higher amount of PC 2‐4 ‐Threshold concentration 20 nM

Andresen et al., 2012, unpublishedhttp://www.chemie.uni‐oldenburg.de/docs/forschungsberichte/fobe9596/3forsch.html

No Cd‐CA in Ceratophyllum?No Cd 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. demersumDistribution of Cd in C. demersum

• µXRF reveals Cd Zn & Mn distribution in Cd• µXRF reveals Cd, Zn & Mn distribution in 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 CadmiumBeneficial effects of Cadmium

Ceratophyllum demersum forms longer lateralCeratophyllum demersum forms longer lateral shoots with 0.01 µg/ml =  ~ 90nM Cadmium

Less Cd than we wanted..

Andresen et al. 2012, unpublishedOrnes & Sajwan, Water, Air, and Soil Pollution 69: 291‐300, 1993.

Distribution of Cd in the oceansDistribution 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 AnhydraseCd Carbonic Anhydrase

‐Zn‐limited Thalassiosira weissflogii growbetter when Cd is added

‐A new CA protein for Cd is expressed‐Cd‐CA larger than Zn‐CACd CA l bi d Z‐Cd‐CA can also bind Zn

‐Cd‐CA activity with Zn slightly, with Cdmuch higher than Zn‐CA

Lane & Morel, PNAS 97(9):4627‐4631, 2000

Cd‐Carbonic AnhydraseCd Carbonic Anhydrase

7 h li 9 β h bi di k‐7 α‐helices, 9 β‐sheets, binding pocket‐ Active site:  Cd bound to 2 Cys, 1 His, 1 H2O‐Tetrahedral conformation

Xu et al., Nature 452:56‐61, 2008

‐ (Acetate as substrate analogue)

Take home messagesTake home messages

• Cadmium can affect a plant in various ways• Cadmium can affect a plant in various ways– Induction of ROS  ROS react with everything in the cellInterfering with PS / respiration / metabolism– Interfering with PS / respiration / metabolism

• Competing with other ions• Replacing other ions in active centres of enzymes• Replacing other ions in active centres of enzymes

• Cadmium can have a metabolic function under certainCadmium can have a metabolic function under certaincircumstances– Replacing other essential but missing ions– Replacing other essential, but missing ions– Concentration dependantHyperaccumulators– Hyperaccumulators

ThanksThanks

Slides on the Küpper group homepage