Heavy Metals and Plants - a complicated relationshipHeavy metal Homeostasis

S h ll kk l i i ildSchwermetall-Hyperakkumulation im Wilden Westenmodified from:

Presented by Hendrik Küpper, Universität Konstanz, at the DAAD summer school 2008

Dose-Response principle for heavy metals

Exclusively toxic metal (e.g. mercury)Mi t i t ( )

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Part I: Heavy metals as micronutrients

a few examples of functions, uptake, transport and deficiency mechanisms

Heavy metal deficiency as a global problem for agriculture

grün = moderate zinc deficiency; rot = severe zinc deficiency

From: Alloway BJ. 2001. Zinc the vital micronutrient for healthy, high-value crops. Brussels, Belgium: International Zinc Association.

Cadmium as a micronutrient

Cadmium as Plant-micronutrient in Thalassiosira weissflogii. A, B: growth of the algae. (Lane and Morel, 2000, PNAS97)

Cadmium deficiency in the Cd/Zn hyperaccumulatorThlaspi caerulescensp

With 10 µM cadmium in the nutrient solution Without cadmium in the nutrient solutionµ--> healthy plants --> damage due to attack of insects

Küpper H, Kroneck PMH (2005) MIBS 44 (Sigel et al., eds), chapter 5

Iron deficiency in the oceans

Iron concentrations at the surface (top picture) and in ( p p )1000m depth (bottom picture)

Source: www-paoc.mit.edu

Mechanisms of Metal Uptake in EucaryotesMain families of metal transport proteinsp p

example: manganese transport in yeast and plants

From: Pittman JK, 2005, NewPhytol167, 733-742

4 main familiesP-type ATPasescation diffusion facilitators (CDF-transporters)cation diffusion facilitators (CDF-transporters)ZRT-/IRT-like proteins (ZIP-transporters)Natural resistance associated Macrophage proteins (Nramp-transporters)

Mechanisms of Metal Uptake in plantsRoot uptake and intracellular distribution in plantsp p

example: iron and zinc transport in Brassicaceae

root uptake

intracellularintracellular distribution

From: Colangelo EP, Guerinot ML, 2006, CurrOpinPlantBiol9:322-330

4 main familiesP-type ATPasescation diffusion facilitators (CDF-transporters)cation diffusion facilitators (CDF-transporters)ZRT-/IRT-like proteins (ZIP-transporters)Natural resistance associated Macrophage proteins (Nramp-transporters)

Mechanisms of Metal Uptake+Compartmentation in Plants (I)CPx-type (=P1B-type) ATPasesCPx-type (=P1B-type) ATPases

Likely Functions (suggested by expression studies)p )

translocation into the root xylem, that means out of root cells ( e.g. HMA4)xylem unloading in shootsxylem unloading in shootsintracellular metal sequestration e.g. in the vacuole (putative!)transport into the chloroplast insidetransport into the chloroplast, inside the chloroplast into the thylakoidstransport into the Golgi apparatus

From: Williams LE, Mills RF, 2005, TrendsPlantSci10, 491-502From: Argüello JM et al., 2007, Biometals,

DOI 10.1007/s10534-006-9055-6

Mechanisms of Metal Uptake+Compartmentation in Plants (I)CPx-type (=P1B-type) ATPasesCPx-type (=P1B-type) ATPases

From: Williams LE, Mills RF, 20052005,

TrendsPlantSci 10,491-502

Sequence characteristics predicted structure(only A P and N domain crystallised from bacterial proteins)(only A, P, and N-domain crystallised from bacterial proteins)

CPx-motif in 6th predicted transmembrane helix ( Name!)Variable number of transmembrane helicesMANY hi tidi d t i i ( 58 C i T HMA4)MANY histidines and cysteines in sequence ( e.g. 58 Cys in TcHMA4)Putative metal binding domain at N-terminus (in cytosol?)Histidine repeat at C-terminus (in cytosol?)

Purification and Characterisation of a Zn/Cd transporting P1Btype ATPase from natural abundance in the Zn/Cd yp

hyperaccumulator Thlaspi caerulescens

From: Aravind P, Leitenmaier B, Yang M, Welte W, Kochian LV, Kroneck PMH Küpper H (2007) BBRC 364 51 56Kroneck PMH, Küpper H (2007) BBRC 364, 51-56

Preliminary characterisation resultspost-translational modification (splitting) possibleregulation by substrate inhibitionregulation by substrate inhibitionhigher activity with Zn with Cd, but higher affinity for Cd than for Zn

Mechanisms of Metal Uptake in Plants (II)ZIP-transportersZIP-transporters

Lik l F ti ( t d bLikely Functions (suggested by expression studies)

uptake of metals into cells over the cytoplasmatic membraneabundant in all eucaryotes, incl. y ,humans, plants and fungi

From:Guerinot ML, 2000, BBA 1465, 190-8

Mechanisms of Metal Uptake+Compartmentation in Plants (II)ZIP-transportersZIP-transporters

Likely Functions (suggested by expression st dies)studies)

• uptake of metals into cells over the cytoplasmatic membrane

From:Guerinot ML 2000 BBA 1465 190 8

• abundant in all eucaryotes, incl. humans, plants and fungi

From:Guerinot ML, 2000, BBA 1465, 190-8

Structure predicted by sequence• usually 8 transmembrane helices, one long

variable region predicted to be in the cytoplasmvariable region, predicted to be in the cytoplasm• 309-476 amino acids

Characteristics revelaed by yeast expression studies

• High affinity and saturable kinetics for selected metal (e.g. Zn in ZNT1)

• Lower affinity uptake for related metals (e.g. Cd inLower affinity uptake for related metals (e.g. Cd in ZNT1)

From:Pence NS et al., 2000, PNAS 97, 4956-60

Mechanisms of Metal Uptake+Compartmentation in Plants (II)Transcriptional regulation of ZIP transporters

Fe deficiency (left) and iron replete conditions (right)

p g p

From:Connolly et al., 2002, PlantCell 14, 1347-57

Expression patternexpressed in most plants mainly under metal deficient conditionsin metal hyperaccumulators rather strong expression at all metal levelsexpression mainly in metabolicallyexpression mainly in metabolically active cells, not metal storage cells

From:Küpper H, Seib LO, Sivaguru M, Hoekenga OA, Kochian LV, 2007 The Plant Journal 50(1), 159-187

Mechanisms of Metal Uptake in Plants (III)Natural resistance associated macrophage proteins (Nramps) atu a es sta ce assoc ated ac op age p ote s ( a ps)

Likely Functions (suggested by expression studies)

Discovered to have a role in the immune response of animals ( name!)b d t i ll t i l habundant in all eucaryotes, incl. humans,

plants and fungipredicted to play a role in uptake into the cell as well as trafficking inside the cell

From: Nevo Y, Nelson N, 2006, BBA 1763, 609-620

Mechanisms of Metal Uptake in Plants (III)Natural resistance associated macrophage proteins (Nramps) atu a es sta ce assoc ated ac op age p ote s ( a ps)

From: Nevo YFrom: Nevo Y, Nelson N, 2006, BBA 1763, 609-

620

Structure prediced from sequence (data from animal proteins)11-12 transmembrane helices, 10 of them with conserveed sequenceqlong loops on both sides of the membranemetal binding sites unclear

Mechanisms of Metal Uptake in Plants (III)Natural resistance associated macrophage proteins (Nramps) atu a es sta ce assoc ated ac op age p ote s ( a ps)

Putative mechanism

proton symport with the electrochemical gradient drives metal translocation against themetal translocation against the gradient – controversial, other studies suggest proton-metal antiport...metal binding induces the proton binding

From: Nevo Y, Nelson N, 2006, BBA 1763, 609-620

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Mechanisms of Metal Uptake in Plants (IV):cation diffusion facilitator (CDF)-Transporterscation diffusion facilitator (CDF)-Transporters

abundant in all eucaryotes, incl. humans, plantshumans, plants and fungidifferent subfamiliessubfamilies

From: Montanini B et al., 2007, BMCGenomics 8, 107

Mechanisms of Metal Uptake in Plants (IV)cation diffusion facilitator (CDF)-Transporterscation diffusion facilitator (CDF)-Transporters

From: Kobae et al., 2004, PlantCellPhysiol 45, 1749-58

From: Blaudez D et al., 2003, PlantCell 15, 2911-28

Functions concluded from expression studies (localisation andFunctions concluded from expression studies (localisation and overexpression/knockout phenotypes)

Metal detoxificationSequester the metals in intracellelular compartments (mainly vacuole)

Mechanisms of Metal Uptake in Plants (IV)cation diffusion facilitator (CDF)-Transporterscation diffusion facilitator (CDF)-Transporters

From: Kobae et al., 2004,

PlantCellPhysiol 45PlantCellPhysiol 45, 1749-58

Sequence characteristics predicted structureMANY histidines in sequence6 transmembrance helicesputative metal binding domain at N-terminus (in cytosol?)likely metal binding histidine repeat in loop (in cytosol?)

Mechanisms of Metal Uptake in Plants (V)long-distance transport ligandslong-distance transport ligands

Histidine

Nicotianamine

Histidine

Nicotianamine

Selected important plant enzymes with iron in their active centrein their active centre

Catalase SOD

Cytochrome cCytb6f

Selected important plant enzymes with copper in their active centrein their active centre

• Plastocyanin

•Multi-copper-oxidases, e.g. laccases

Selected important plant enzymes with zinc in their active centre

Carboanhydrasein their active centre

Zinc finger-motive

Tyrosin phosphatase

Metal efficiencyexample: zinc

From: Hacisalihoglu G, Kochian, LV.How do some plants tolerate low levels of soil zinc? M h i f i ffi i i l tMechanisms of zinc efficiency in crop plants.

New Phytologist 159 (2), 341-350.

Part II: Heavy metal toxicity

Environmental relevance of heavy metal toxicity

A seemingly intact, natural creek ...However, the Elodea canadensiso e e , t e odea ca ade s sinside died from zinc stress that converted its chlorophyll to Zn-chlorophyllp y

Zn-Fluosilicate

Küpper H, Küpper F, Spiller M (1996) Journal of Experimental Botany 47 (295), 259-66

Environmental relevance of heavy metal toxicity

Where? How? Why?

• Naturally on heavy metal rich soils(Cu: e.g. in Zaire, Afrika; Zn/Cd: rel. frequent, incl. Europe; Ni: rel frequent serpentine soils e gEurope; Ni: rel. frequent, serpentine soils e.g. in Africa, Australia, North and Middle Amerika): Heavy metal concentrations high enough for being toxic for most organisms.

• Naturally in copper-rich areas of the oceans (e.g. Sargasso sea): Cu-concentrations in the nanomolar range already inhibit some sensitive cyanobacteria.

• Anthropogenically due to the use of heavy metal salts (e.g. CuSO4, z.B. Zn-phosphid, Zn-borate, Zn-fluosilicate): concentrations in themicromolar range are toxic for many plants, mainly water plants in neighbouring ponds g y p , y p g g pand creeks

• Anthropogenically due to ore mining and refining, concentrations in the vicinity of mines, smelters and rubble dumps can be extremely high and toxic for all organisms.

• Anthropogenically due to the activities of other inductries. The longest river in Germany the Rhine contained up to 0 5 µM copper in the 1970‘s which is lethal forGermany, the Rhine, contained up to 0.5 µM copper in the 1970‘s, which is lethal for sensitive water plants like Stratiotes or Elodea.

1. Inhibition of root function

Why roots?

• In terrestrial plants the root is generelly the first organ that comes into contact with the heavy metals.

• In the case of heavy metals with typically low mobility, e.g. copper, also the highest metal accumulation is found in the roots

1. Inhibition of root function

From: Küpper H, Kochian LV, 2008,

unpublishedunpublished

Mechanisms

• Competition in the uptake of less available essential micronutrients, which are sometimes transported by the same proteins

• Enhanced precipitation of essential micronutrients at the root surface

• Inhibition of transport proteins?

• Diverse relatively unspecific inhibitions of cytoplasmic enzymes

• Inhibition of cells division (relevance and mechanism unclear!)

• As a result of roots toxicity, roots tips and root hairs die off

Genotoxicity Micronucleus (MCN) formation

Relevance

Strongly DEPENDS on the metal applied:

• NOT relevant for copper and zinc toxicity, because other mechanisms (mainly photosynthesis inhibition) are MUCH morephotosynthesis inhibition) are MUCH more efficient

• VERY relavant for cadmium, because ,genotoxicity is very efficient, comparable e.g. to photosynthesis inhibition

• For lead, it is not very efficient, but other mechanisms are even less efficient because the metal is generally NOT very toxic for plants! g y y p

Pb toxicity in general NOT environmentally relevant !

Also depends on the plant species!

From: Steinkellner H, et al., 1998, Env.Mol.Mutag. 31, 183-191

Genotoxicity

Mechanisms: Point Mutations and Homologous recombinations

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From: Kovalchuk O, Titov V, Hohn B, Kovalchuk I, 2001, Nature Biotechnol. 19, 568-72

Genotoxicity

Mechanisms: Mitotic aberrations induced by phenyl mercuric acetate (PMA)

Micronucleus (MCN) formationAbnormal spindle

C-metaphase Tripolar anaphase

Star anaphaseAnaphase with a pair of lagging chromosomesStar anaphase lagging chromosomes

Micronucleus (MCN) formationPMA concentration (ppm) PMA concentration (ppm)

From: Dash S, Panda KK, Panda BB, 1988, Mutation Research 203, 11-21

2. Oxidative StressR lRelevance

• NOT clear: Studies with environmentally relevant realistic but still toxic heavy metal concentrations oft do NOT show oxidative stress! Almost all studies concluding thatconcentrations oft do NOT show oxidative stress! Almost all studies concluding that oxidative stress would be a major factor in heavy metal induced inhibition of plant metabolism were carried out using extremely high metal concentrations.

Mechanisms generating reactive oxygen species during heavy metal stress

• Direct: catalysed by redox-active metal ions (Fe2+, Cu+), hydrogen peroxide is converted to reactive oxygen radicals via the Fenton-Reaction:

• Indirect: malfunktion of photosynthesis and respiration can generate reactive oxygen species Therefore even in vivo redox inert Metal ions like Zn2+ and Cd2+ can causespecies. Therefore, even in vivo redox-inert Metal ions like Zn2+ and Cd2+ can cause oxidative stress.

2. Oxidative Stress

Mechanisms of damage caused by oxidative stress in plants

•Oxidative stress can lead to oxidation of Lipids in•Oxidative stress can lead to oxidation of Lipids in membranes and thus make them leaky. This is a popular but debated mechanism.

• Oxidation of proteins

From: en.wikipedia.org

3. Environmental relevance of heavy metal induced inhibition of photosynthesis:

100 100

heavy metal induced inhibition of photosynthesis: Elodea stressed by 0.2 µM (= 0.013 ppm) Cu2+

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Küpper H, Küpper F, Spiller M (1996) Journal of Experimental Botany 47 (295), 259-66

3. Heavy metal induced inhibitions of photosynthesis:suggested targets Details in separate lecture...suggested targets Details in separate lecture...

heavy metals

P700* A0

heavy metals

A0 A1

P680* Ph

Antenna Chl-protein

l P680 PheoQA QB ChlChlPQ FeS/

complexes,main protein:

LHCII4 h·νE

ChlRieske

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electron transport

4 h ν

ChlP700

excitation energy

ChlChl

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2 H2OWSC 4 e