Heavy Metals and Plants - a complicated relationship Heavy metal detoxification

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1 Heavy Metals and Plants - a complicated relationship Heavy metal detoxification Schwermetall-Hyperakkumulation im Wilden Westen modified from: Presented by Hendrik Küpper, Universität Konstanz, at the DAAD summer school 2008

2 Dose-Response principle for heavy metals Küpper H, Kroneck PMH, 2005, Metal ions Life Sci 2, 31-62

3 Cd-acclimation in Thlaspi caerulescens Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175,

4 1. General Resistance-Mechanisms 1.1. Heavy metal detoxification with strong ligands Phytochelatins Bind Cd 2+ with very high affinity, but other heavy metal ions with low affinity Specially for Cd 2+ -binding synthesized by phytochelatin-synthase They are the main Cd-resistance mechanism in most plants (except hyperaccumulators) and many animals Phytochelatin synthase becomes activvated by (e.g. via Cd-binding) blocked thiols of glutathion and similar peptides Phytochelatins bind Cd 2+ in the cytoplasm, then the complex is sequestered in the vacuole. In the vacuole large phytochelatin-cd-aggregates are formed

5 1. General Resistance-Mechanisms 1.1. Heavy metal detoxification with strong ligands Glutathion Also glutathione itself, the building block of phytochelatins, can bind and thus detoxify heavy metals - the in vivo relevance is questionable Metallothionins MTs of type I und II bind Cu + with high affinity and seem to be involved in its detoxification. BUT: Main role of MTs in plants seems to be Metal-distribution during the normal (non-stressed) metabolism

6 1. General Resistance-Mechanisms 1.1. Heavy metal detoxification with strong ligands Free Amino acids Like during metal transport under normal physiological conditions, free amino acids (mainly histidine+nicotianamine) seem to play a role in the detoxification of heavy metals. Histidine Nicotianamine

7 Other Ligands Diverse Proteins 1. General Resistance-Mechanisms 1.1. Heavy metal detoxification with strong ligands Anthocyanins: seem to be involved in Brassicacae in Molybdemum binding (detoxification or storage?) Hale et al_2001, PlantPhysiol 126, Cell wall Carr HP, Lombi E, Küpper H, McGrath SP, Wong MH (2003) Agronomie 23, Some algae release unidentified thiol-ligands during Cu-stress

8 4.2. Heavy metal detoxification by compartmentation Mechanisms Generelly: aktive transport processes against the concentration gradient transport proteins involved. Exclusion from cells: - observed in brown algae - in roots Sequestration in the vacuole: Küpper H et al., 2001, J Exp Bot 52 (365), plant-specific mechanism (animals+bacteria usually don t have vacuoles...) - very efficient, because the vacuole does not contain sensitive enzymes - saves the investment into the synthesis of strong ligands like phytochelatins - main mechanism in hyperaccumulators Sequestration in least sensitive tissues, e.g. the epidermis instead of the photosynthetically active mesophyll Küpper H, Zhao F, McGrath SP (1999) Plant Physiol 119,

9 4.3. Further Resistance-Mechanisms Reduction by reductases, e.g. Hg 2+ --> Hg 0, Cu 2+ --> Cu + Rugh CL, et al, 1996, PNAS 93, Precipitation of insoluble sulfides outside the cell (on the cell wall) Methylation, e.g. of arsenic

10 1. Root-specific resistance mechanisms Zn-sensitive Strategies Reduction of the unspecific permeability of the root for unwanted heavy metals: expression of peroxidases enhances lignification Active (ATP-dependent) discharge by effluxpumps: was shown for Cu in Silene vulgaris (and for diverse metals in bacteria). Zn-resistant Chardonnens AN, Koevoets PLM, vanzanten A, Schat H, Verkleij JAC, 1999, PlantPhysiol120_

11 3. Resistance mechanisms against oxidative stress Enhanced expression of enzymes that detoxify reactive oxygen species (superoxide dismutase+catalase. Problem: inhibition of Zn-uptake ( SOD) during Cd-Stress. Synthesis of non-enzyme-antioxidants, e.g. ascorbate and glutathione Changes in the cell membranes to make them more resistant against the attack of reactive oxygen species: - Lipids with less unsaturated bonds - Exchange of phosphatidyl-choline against phosphytidyl-ethanolamine as lipid- head - Diminished proportion of lipids and enhanced proportion of stabilising proteins in the membrane

12 Part II: Plants with an unusual appetite: Heavy metal hyperaccumulation Shoot dry weight (g) (a) 14 Thlaspi goesingense Alyssum bertolonii 12 Alyssum lesbiacum Ni concentration (µg g -1 ) (b) Thlaspi goesingense Alyssum bertolonii Alyssum lesbiacum Ni added to the substrate (mg kg -1 ) Ni added to the substrate (mg kg -1 ) Effects of Ni 2+ addition on hyperaccumulator plant growth and Ni 2+ concentration in shoots Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP (2001) J Exp Bot 52 (365),

13 Cadmium deficiency in the Cd/Zn hyperaccumulator Thlaspi caerulescens demonstrates the biological function of hyperaccumulation... With 10 µm cadmium in the nutrient solution --> healthy plants Without cadmium in the nutrient solution --> damage due to attack of insects Küpper H, Kroneck PMH (2004) MIBS 44 (Sigel et al., eds), chapter 5

14 How do hyperaccumulators bind heavy metals? Analysis of metal ligands by Extended X-ray Absorption Fine Structure (EXAFS)

15 Speciation of cadmium and zinc hyperaccumulated by Thlaspi caerulescens (Ganges ecotype) Analysed by X-ray absorption spectroscopy of frozen-hydrated tissues Fourier Transform of χ*k Zn Cd histidine contributio n increase in sulphur contribution young leaves mature leaves mature stems young leaves senescent leaves mature stems Distance [Å] Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2003) Plant Physiology 134 (2),

16 Speciation of cadmium hyperaccumulated by Thlaspi caerulescens (Ganges ecotype) Analysed by X-ray absorption spectroscopy of frozen-hydrated tissues Percent of all ligands binding to Cd young mature senescent dead 0 stems petioles leaves Developmental stage of leaves Tissue sulphur ligands N/O ligands Percent of all ligands binding to Cd Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2003) Plant Physiology 134 (2),

17 Speciation of cadmium and copper in the Cu-sensitive CdZn-hyperaccumulator T. caerulescens Analysed by XAS of frozen-hydrated tissues Cu-K-edge EXAFS Cd: Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2003) Plant Physiology 134 (2), Cu: Mijovilovich A, Meyer-Klaucke W, Kroneck PMH, Küpper H - unpublished

18 Where do hyperaccumulators store metals? Measurements by Energy Dispersive X-ray Analysis (EDXA) peaks of characteristic x-ray photons spectral window Counts continuous background of bremsstrahlung Energy / kev Analysis: a) recording of complete spectrum, subtraction of background --> quantification of peak areas by comparison to internal standard b) recording of counts in spectral window --> dot maps, line scans

19 mm / mm Ca 8 young leaves Compartmentation of metals in leaves Zn/Cd/Ni accumulation in epidermal vacuoles of Thlaspi and Alyssum species epidermis mesophyll upper lower vacuole 0 8 Zn Mg P S Cl K mature leaves mm / mm Ca Zn Mg P S Cl K upper epidermis upper mesophyll lower mesophyll lower epidermis Concentrations of elements in leaf tissues Thlaspi caerulescens Zn K α line scan and dot map of a T. caerulescens leaf Ni K α line scan and dot map of a A. bertolonii leaf Zn: Küpper H, Zhao F, McGrath SP (1999) Plant Physiol 119, Ni: Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP (2001) J Exp Bot 52 (365),

20 Compartmentation of metals in shoots Subcellular localisation of Ni in the epidermis of Thlaspi goesingense

21 Compartmentation of Zn and Ni in leaves Functional differenciation of epidermal cells of Thlaspi species Linear Regression: Y = A + B * X 8 6 Parameter Value Error A B R SD N P mm Zn / mm Ca data points regression line confidence limits cell width Upper leaf surface of T. goesingense Correlation between cell size and zinc concentration Dot map of the Ni K alpha line Zn: Küpper H, Zhao F, McGrath SP (1999) Plant Physiol 119, Ni: Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP (2001) J Exp Bot 52 (365),

22 Compartmentation of metals in leaves Zn/Cd accumulation in epidermal trichomes of Arabidopsis halleri Küpper H, Lombi E, Zhao FJ, McGrath SP (2000) Planta 212, 75-84

23 Compartmentation of elements in leaves: Correlation between metals in the mesophyll ofarabidospis halleri Cd / mm 6 4 Mg / mm data points regression line (R = 0.94; P < ) 95% confidence limits Zn / mm 5 0 data points regression line (R = 0.79; P < ) 95% confidence limits Cd / mm Küpper H, Lombi E, Zhao FJ, McGrath SP (2000) Planta 212, 75-84

24 Cd-transport into protoplasts isolated from the hyperaccumulator plant Thlaspi caerulescens revealed by in vivo fluorescence kinetic microscopy Transmitted light: information about structure and cell type Rhod5N fluorescence: cadmium measurement Leitenmaier B, Küpper H, (2008) unpublished

25 Cd-transport into protoplasts isolated from the hyperaccumulator plant Thlaspi caerulescens... (II) In almost all measured cells, a bright cytoplasmatic ring appeared first after start adding Cd to the medium. A cell that was incubated with Cd over night is completely filled with Cd, which means that the transport into the vacuole took place The transport into the vacuole is the time-limiting step in metal uptake! Leitenmaier B, Küpper H, (2008) unpublished

26 Cd-transport into protoplasts isolated from the hyperaccumulator plant Thlaspi caerulescens...(iii) higher uptake rates in large metal storage cells compared to other epidermal cells are caused by higher transporter expression, NOT by differences in cell walls or transpiration stream Leitenmaier B, Küpper H, (2008) unpublished

27 Regulation of ZNT1 transcription analysed by quantitative mrna in situ hybridisation (QISH) in a non-hyperaccumulating and a hyperaccumulating Thlaspi species µm Zn 2+ Thlaspi caerulescens 10 µm Zn 2+ Thlaspi arvense 1 µm Zn 2+ Thlaspi arvense c(znt1 mrna) / c(18s rrna) spongy mesophyll phloem bundle sheath palisade mesophyll epidermal subsidiary cells epidermal guard cells epidermal metal storage cells Küpper H, Seib LO, Sivaguru M, Hoekenga OA, Kochian LV (2007) The Plant Journal 50(1),

28 Quantitative cellular pattern of ZNT5 transcript abundance in young leaves of Thlaspi carulescens (Ganges ecotype) judged by its expression pattern in the epidermis, ZNT5 may be a key player in hyperaccumulation of Zn Küpper H, Seib LO, Kochian LV - unpublished

29 Regulation of ZNT5 transcription in young leaves of Thlaspi carulescens (Ganges ecotype) analysed by QISH Küpper H, Seib LO, Kochian LV - unpublished ZNT5 seems to be involved both in unloading Zn from the veins and in sequestering it into epidermal storage cells

30 Purification and Characterisation of a Zn/Cd transporting P1B type ATPase from natural abundance in the Zn/Cd hyperaccumulator Thlaspi caerulescens Scheme from: Solioz M, Vulpe C 1996) TIBS21_ Aravind P, Leitenmaier B, Yang M, Welte W, Kochian LV, Kroneck PMH, Küpper H (2007) BBRC 364, post-translational modification of TcHMA4 K D of TcHMA in the high nanomolaar to low micromollar range

31 UV- Vis-Spectroscopy of TcHMA4 Ligand-metal charge-transfer transitions of Cd-cysteine bonds Leitenmaier B, Meyer-Klaucke W, Aravind P, Kroneck PMH, Küpper H (2008) unpublished

32 EXAFS-analysis of TcHMA4 and another, so far unknown Cd-ATPase First shell mainly S First shell mainly S, evidence for Cd-S-cluster from multiple scattering Leitenmaier B, Meyer-Klaucke W, Aravind P, Kroneck PMH, Küpper H (2008) unpublished

33 Compartmentation of elements in leaves: Correlation between metals and cells with differing physiology in the mesophyll of metal-stressed hyperaccumulators Cd / mm Mg / mm data points regression line (R = 0.94; P < ) 95% confidence limits Zn / mm data points regression line (R = 0.79; P < ) 95% confidence limits Cd / mm Cd vs. Zn, Cd vs. Mg: Küpper H, Lombi E, Zhao FJ, McGrath SP (2000) Planta 212, Ni vs. Mg: Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP (2001) J Exp Bot 52 (365), Heavy metal chlorophylls: Küpper H et al JExpBot, 1998 PhotRes, 2002 JPhycol, 2003 FunctPlantBiol

34 Cd-stress in the Zn-/Cd-hyperaccumulator T. caerulescens: distribution of photosystem II activity parameters Control 30 C D T. caerulescens Control A 0 20 B T. fendleri Cellular F v /F m distribution in a control plant Stressed E Stressed 10 0 Acclimating F in a Cd- Distribution of F v /F m stressed plant Acclimated F v / F m Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, transient heterogeneity of mesophyll activity during period of Cd-induced stress

35 Cd-stress in the Zn-/Cd-hyperaccumulator T. caerulescens: correlation between PSII activity parameters and Cd-accumulation F v /F m Fluorescence of Cd-dye transient heterogeneity of mesophyll activity during period of Cdinduced stress correlates with transient heterogeneity of Cdaccumulation! Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175,

36 Proposed mechanism of emergency defence against heavy metal stress Normal: Sequestration in epidermal storage cells Stressed: additional sequestration in selected mesophyll cells Acclimated: Enhanced sequestration in epidermal storage cells v v v Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175,

37 Phytoremediation comparison of species plant species max. Cd Biomass Cd-removal mg/kg DW t DW/ha g/(ha*year) Arabidopsis halleri Thlaspi caerulescens (Prayon) Thlaspi caerulescens (S. France) Dichapetalum gelonoides Athyrium yokosense Arenaria patula Sedum alfredii Poplar/Pappel Upland Rice Data from field experiments, carried out by Rufus Chaney (USA), presented in Hangzhou 2005 hyperaccumulators are, despite their low biomass, the best-suited plants for phytoremediation!

38 Application of hyperaccumulators fro phytomining Vegetation on naturally nickel-rich soil (Serpentine). Such soil is neither usable for agriculture (Ni-concentration far too high) nor for conventional ore mining (Ni-concentration too low). Nickel-hyperaccumulators on such soils enrich the Ni to several percent of their shoot dry mass. After burning them, the ash contains 10 to 50% Ni, so that it can be used as a bio-ore. Phytomining pictures from R. Chaney Such a plant mine can, according to field studies under commercial conditions, yield around 170 kg Ni per hectare and year. At the current (average Jan-May 2008) Ni price of around 18 per kg raw nickel these are about 3000 per hectare and year.

39 Phytoextraction in action The location: a base-metal smelter, South Africa The problem: Ni contamination over 5ha due to Ni salt storage and spillage The solution: phytoextraction using a native nickel-accumulating species From: Presentation of Chris Anderson at CERM3 meeting

40 Interactions of plants with potentially toxic metals complexation by strong ligands (e.g. phytochelatins) metal-induced damage to photosynthesis (e.g. Mg-substitution) complexation by strong ligands (e.g. phytochelatins) oxidative stress and other damage cytoplasm vacuole vacuole cytoplasm cell walls epidermis cells (not present e.g. in Elodea) photosynthetic cells (e.g. mesophyll cells, stomatal guard cells) cell walls stem epidermis regulation by boundary cells? leaf phloem stem phloem leaf xylem stem xylem ± root to shoot translocation in the case of submerged aquatic plants cell walls vacuole root root uptake exclusion mechanisms damage to roots Heavy metals in soil or water