This document discusses heavy metal tolerance in plants. It provides information on nickel hyperaccumulators like Sebertia acuminata that can contain 2.5% nickel in its leaves. It also mentions Arabidopsis arenosa, an annual herb that shows tolerance to zinc, lead and cadmium. The document covers topics like the definition and characteristics of heavy metals, their toxicity mechanisms in plants, and the various tolerance strategies plants have evolved, including avoidance, tolerance, sequestration and hyperaccumulation.

2. [http://www.bbg.org/]
Sebertia acuminata is a nickel hyperaccumulator; it contains approximately
2.5% nickel in its leaves.
(Perrier et al. 2004)
The total nickel content of a single mature tree was estimated to be 37 kg.
(Sagner et al. 1998)
Slota M. 2013 - 2 -INTRODUCTION

3. Arabidopsis arenosa is an annual, biennial or short-lived perennial herb.
Former studies have shown a wide range of its constitutive tolerance to zinc,
lead and cadmium in calamine populations and the presence of adaptations
to zinc hyperaccumulation (Reeves et al. 2001).
The population of A. arenosa on the zinc smelter tailings heap in Miasteczko Śląskie. Phot. M. Slota.
Slota M. 2013 - 3 -INTRODUCTION

4. Heavy metals – definition, origin and characterization.
Heavy metals molecular action and toxicity.
Mechanisms of heavy metal tolerance in plants.
Genetic basis of heavy metal tolerance.
Evolution of heavy metals tolerance.
Slota M. 2013 - 4 -INTRODUCTION

5. [www.wps.prenhall.com]
Ten most abundant elements by
mass in the Earth's crust (A) and in
living organisms (B).
Slota M. 2013 - 5 -
A visualization of periodic table
that aims to assort the elements
according to their relative
abundance at the Earth’s
surface. »
A. B.
[http://www.periodictable.com]
10-8 10-6 10-4 0,01 1Abundance [%]
HEAVY METALS – GENERAL CHARACTERISTICS

6. Summary of the frequency of metals in the centers of active enzyme in eukaryotes
(Waldron et al. 2009, modified).
The height of each column and presented values ​​determine the percent of known enzymes
characterized by the presence of the metal.
Slota M. 2013 - 6 -
3-D model of the zinc finger motif
characteristic for metalloproteins.
The zinc ion is highlited green. »
HEAVY METALS – GENERAL CHARACTERISTICS

7. Slota M. 2013 - 7 -
Knowledge of the toxicity of heavy metals has been based on research carried
on limited number of model species.
➢ Occasionally, new studies on endemic species indicates the occurrence of
alternative ion homeostasis patterns.
➢ The metabolic pathways of specific organisms adapted to metal contrentations
which are generally considered to be toxic at any concentration.
➢ Marine diatom ,Thalassiosira weissflogii hpossess an alternative version of the
carbonic anhydrase enzyme, (CD-CA), in which the Zn2+ metallic cofactor is
replaced with cadmium Cd2+ (Park et al. 2008).
« Scanning electron
micrograph of the
T. weissflogii cells.
3-D structure of a single repeat of CDCA
(Cd-carbonic anhydrase). »
[http://cmore.soest.hawaii.edu] [http://www.princeton.edu]
Cd2+ ion
HEAVY METALS – GENERAL CHARACTERISTICS

8. Periodic table distinguishing essential from toxic elements as well as those
considered as both essential and toxic.
(Gomes and Silva 2007)
Slota M. 2013 - 8 -HEAVY METALS – GENERAL CHARACTERISTICS

9. MACROELEMENTS
MICROELEMENTS
BIOGENIC ELEMENTS
TOXIC METALS
Fe, Cu, Co, Cl, Mo,
Mn, Zn, Ni, B
K, Ca, Mg, P, S, N
C, O, H, N, P, K, Ca, S, Mg
(>0,1% plant dry weight)
Cd, Pb, Sn, Hg
Slota M. 2013 - 9 -
Theoretical models of the dose-response reaction in biological systems for different
groups of metals (A - microelements, B - macroelements, C - toxicants).
(Wright, Welbourn 2002, modified).
DOSE
STIMULATIONINHIBITION
HEAVY METALS – GENERAL CHARACTERISTICS
METAL
METAL CONCENTRATION [mg/kg]
DEFICIT OPTIMAL TOXIC
Zinc (Zn) <20 20-150 >400
Manganese (Mn) <20 20-250 >500
Copper (Cu) <40 5-20 >40
Iron (Fe) <50 50-250 >500
Reference levels of deficient, physiological and toxic metal concentrations of
selected metals (Schulze et al. 2005, modified).

10. HEAVY METALS are a members of a loosely defined subset of
elements that exhibits metallic properties and exceed a border
density value of 4,5 (5) g/cm3 and show a tendency in chemical
reactions to donate electrons to form a simple cations.
The term 'heavy metal' because of its inaccuracy is controversial
and is often replaced by the term toxic metals (Duffus 2002).
Localization of defined groups of chemical elements due to
its redox properties within the periodic table (Bishop 2001). »
Slota M. 2013 - 10 -HEAVY METALS – GENERAL CHARACTERISTICS

11. (Reichman 2002)
The slowdown in growth,
reduced plant biomass,
reduced root system.
Increased lignification and
calose deposition in the
cell walls, chlorosis,
necrosis and leaf
distortion.
Decline in the efficiency of
photosynthesis by chlorophyll
synthesis disorders, loss of
water and signs of wilting.
Changes in the physicochemical
properties of cell membranes, disrupted
water uptake by the roots and inhibition
in other minerals uptake.
Morphological symptoms
Physiological symptoms
Enzymatic symptoms
Dysfunction of
photosynthetic,
respiratory chain and the
Krebs cycle enzymes.
Nitrogen and phosphorus
metabolism disorders.
Increase in formation of
reactive oxygen species.
Slota M. 2013 - 11 -MECHANISMS OF TOXICITY

12. ENZYMATIC
DISORDERS
•
CELL MEMBRANE
REACTIVE OXIGEN
SPECIES FORMATION
(Reichman 2002)
▪ Modification of the
biophysical properties of
the cell membranes.
▪ Changes in
permeability.
▪Generation of excess
ROS that are sources
of oxidative stress.
▪ Peroxidation of cell
components.
▪ Competitive displacing
of native ions of the
active sites of enzymes.
▪ Blocking of the enzyme
functional groups
(SH, COOH).
Ag, As, Hg, Pb, Sb, W Ag, Au, Br, Cd, Cu, Hg, Pb Fe, Cu, Cd, Hg, Pb, Zn
Slota M. 2013 - 12 -MECHANISMS OF TOXICITY

13. Steric similarity of
toxic metals and
native ions from
sites of enzymes.(Ashraf et al. 2010)
ENZYMATIC
DISORDERSChlorophyll
synthesis
Photosynthesis
Nitrogen
metab.
Sugar
metab.
Antiox.
processes
Slota M. 2013 - 13 -MECHANISMS OF TOXICITY

14. Avoidance
Tolerance
STRATEGIES FOR ADAPTATION OF PLANTS TO EXPOSURE TO HEAVY METALS
(Hall, 2002)
early reproduction
shortening of life cycle
functioning of metal
excretion channels
presence of
protective barriers
intracellular chelation
chelation in the
rhizosphere
metal hyperaccumuation
cell wall modification
[www.food4livecells.com]
sequestration in vacuole
Slota M. 2013 - 14 -TOLERANCE STRATEGIES

15. STRATEGIES FOR ADAPTATION OF PLANTS TO EXPOSURE TO HEAVY METALS
Slota M. 2013 - 15 -TOLERANCE STRATEGIES
(Prasad and Strzałka, 2002)
Soil metal content
Plantmetalcontent
Hyperaccumulation
Accumulation
Indication
Exclusion

16. Slota M. 2013 - 16 -
(Clemens et al. 2002, modified)
TRAFFICKING AND
SEQUESTRATION
XYLEM
TRANSPORT
MOBILIZATION UPTAKE AND SEQUESTRATION
UNLOADING AND
SEQUESTRATION
Molecular mechanisms
proposed to be involved in
transition metal accumulation
by plants.
CW - cell wall; M - metal; filled circles -
chelators; filled ovals - transporters;
ellipses – metallochaperones.
MECHANISMS OF ADAPTATION

17. Slota M. 2013 - 17 -MECHANISMS OF ADAPTATION
CELLULAR MECHANISMS OF HEAVY METAL TOLERANCE
Exudates
Ectomycorrhiza
Phytochelatins,
organic acids
Nucleus
Vacuole
Cytoplasm
Cell wall
Cell membrane
Expression
ACTIVE
EXCLUSION
OF METALS
PHYSIOLOGICAL
RESPONSE
PROTECTIVE
BARRIERS
SEQUESTRATION
(Hall 2002,
modified)

18. outer
inner
IRT1,IRT3 ZIP 1-5 ZNT1 NRAMP COPT1 NtCBP4 LCT1
X X
X X X X X
X X
X X
X X
X X X X
Fe
Zn
Mn
Ni
Pb
Cd
Slota M. 2013 - 18 -
Plant-specific metal transporting proteins localized in cell membrane participating
in the ion sequestration. The effect of excessive concentration metal in a cell is
increased expression of transporter genes involved in its sequestration accompanied
by a decrease in activity of the main conveyor involved in the uptake rate.
MECHANISMS OF ADAPTATION

19. Slota M. 2013 - 19 -
GROUP TRANSPORTER
TRANSPORTED METAL
FUNCTION
SPECIFIC NONSPECIFIC
ZIPfamily
IRT1 Fe2+ Mn2+, Zn2+, Co2+ uptake
IRT2 Fe2+ uptake
AtIRT3 AtZIP15, AtZIP912,
AtZIP13
Zn2+ uptake
ZIP4, ZIP10, IRT3 Zn2+ uptake (hyperaccumulators)
ZNT1 Zn2+ Cd2+ uptake and transport
NR
AM
P
AtNRAMP1 Fe2+ Mn2+, Zn2+, Cd2+ uptake
TjNRAMP4 Ni2+ transport to protoplast
ATPases
typeP
HMA1-HMA4 Zn2+, Cd2+, Pb2+, Co2+
uptake AtHMA3- metal
sequestration
HMA5-HMA8 Cu2+, Ag2+ cytozol detoxification
AtHMA2, AtHMA4 Zn2+ Cd2+
translocation to shoot (xylem,
phloem), cytozol detoxification
ABC
AtMRP3, AtATM3, AtPDR8,
AtPDR12
Cd2+, Pb2+ cytozol detoxification
HMT1 Cd2+
equestration of Cd2+-
phytochelatin complexes
CDF COPT1-COPT5 Cu2+ uptake
MTP1-3 Zn2+ sequestration in vacuole
IREG CAX1, CAX2 Ca2+ Cd2+ sequestration in vacuole
LCT1 Ca2+, Cd2+, Pb2+, Na+, K+ transport, cytozol detoxification
Transport systems of metal ions in plant cell.
(Woźny and Goździcka-Józefiak 2010, modified)
MECHANISMS OF ADAPTATION

20. Phytochelatins
Metallothioneins
AminoacidsOrganic acids
Phenolic
compounds
Among the primary mechanisms of the neutralization of heavy metal ions to be
bound by the negatively charged chelating agents. The metal complexes are then
deposited in the vacuole or cell wall to neutralize its toxicity.
Metallothionein (MT) and phytochelatins are a families of cysteine-rich, low
molecular weight proteins.. MTs have the capacity to bind both physiological (such
as zinc, copper, selenium) and xenobiotic (such as cadmium, mercury, silver,
arsenic) heavy metals through the thiol group of its cysteine residues,
Slota M. 2013 - 20 -
« Synchrotron micro-XRF (SXRF)
images of a leaf edge from the Ni
hyperaccumulator Alyssum murale.
MECHANISMS OF ADAPTATION
[http://www.regional.org.au/]

21. Slota M. 2013 - 21 -
GROUP GENE PRODUCT FUNCTION
ATPases type P AtHMA1-6, RAN1, PAA1 Zn and Cd transport, Cu transport to chloroplasts
NRAMP family
transporters
AtNramp1, 3, 4 Ca homeostasis (nonspecific Cd uptake)
CDF family
trensporters
ZAT1, ZTP1, TgMTP1 Zn sequestration
ABC type
transporters
AtMRP Cd and Pb transport to vacuole
Antiporters AtMHX1 Mg/Zn antiporter
ZIP transporters
IRT1, ZIP 1-4, ZNT1, 2,
MtZIP2
Fe homeostasis. Zn uptake
(less specific Zn and Cd uptake).
Other tranporters
COPT1, LCT1, AtDX1,
NtCBP4
Cu, Rb, Na, Ca, Cd uptake
Metallothioneins MTF1 metal binding inside cytoplasm
Phytochelatins AtPCS1 metal binding inside cytoplasm
Organic acids - metal binding inside cytoplasm
The list of factors involved in plant metal tolerance (Hassinen et al. 2007, modified).
MECHANISMS OF ADAPTATION

22. Slota M. 2013 - 22 -METAL HYPERACCUMULATION
Transcriptional responses of
selected candidate genes to Zn
and other ions. Transcript levels
relative to EF1α were assessed
in roots (A) and shoots (B) of
hydroponically grown plants
subjected to short-term
treatment with high Zn, long-term
Zn deficiency, or short-term
treatment with Cd, Cu, or Na.
(Talke et al. 2006)

23. cytosol
vacuole
+ organic
acids
SEQUESTRATION
LEAF CELL
METAL UPTAKE SYSTEM
ZIP Nram
p
LC
T
IR
T
HMA
Fe, Cd Ca, Zn,
Mn, Cd, Pb
Zn, Cd Zn, Cd,
Pb, Co
Fe, Cd
ZIP
ROOT CELL
MTP
BOUND METAL
TRANSPORT
+ histidine,
nikotianamine
Hyperaccumulation-associated processes.
(Verbruggen et al. 2008, modified).
Slota M. 2013 - 23-METAL HYPERACCUMULATION

24. DICOTYLEDONES MONOCOTYLEDONES FERNS BRYOPHYTES ALGAE
METAL TOLERANCE
EVOLUTION LINEAGES
Chlorella sp.
Coenochloris sp.
Stichococcus sp.
Mielichhoferia sp.
Dryptodon sp.
Scopelphila sp.
Crumia sp.
Gymnocolea sp.
Ditrichum sp.
Pteris vittata
Pellaea falcata
Athyrium sp.
Dennstaedtia sp.
Doodia sp.
Hypolepis sp.
Nephrolepis sp.
Arabidopsis sp.
Brassica sp.
Thlaspi sp.
Alyssum sp.
Silene sp.
Viola sp.
…
Fetuca rubra
Festuca lemanil
Schoenus nigricans
Deschampsia sp.
Agrostis sp.
(Shaw 2000)
Slota M. 2013 - 24 -EVOLUTION OF METAL TOLERANCE

25. Slota M. 2013 - 25 -EVOLUTION OF METAL TOLERANCE
METAL ORDER SPECIES
Cd, Co, Cu, Ni, Zn
Brassicales, Caryophyllales,
Plumbaginales, Poales
Thlaspi caerulescens, Silene vulgaris,
Armeria maritima, Agrostis tenuis
Co, Cu, Ni
Asterales, Commelinales,
Cyperales, Ericales, Fabales,
Lamiales, Liliales
Inula germanica, Ononis spinosa, Marrubium
vulgare, Anagalis tenella Allium
ampeloprasum
Cd, Pb, Zn Fagales Myrica gale
Cd, Zn Rosales, Malpighiales, Violales Sedum alfredii, Viola calaminaria
Characteristic trends in the distribution of adaptations to metal tolerance within
various orders of angiosperms (Ernst in 2006, modified).
Phylogenetic distribution of species possesing acquired adaptations to metal
tolerance among taxonomic groups is unequal (Ernst 2006).
There is a noticable trend within the different plant orders to differential tolerance to
specific metals. Among taxonomic groups of plants that possessed a set of
specialized adaptations determining high level of tolerance to metals, this feature is
particularly strongly representated of members of the family Brassicaceae
(Peer et al. 2006).

26. Slota M. 2013 - 26 -POSSIBLE APPLICATIONS
(Van Aken and Geiger, 2010)
Phytoremediation is defined as the use of vascular plants to remove or mitigate
the impact of pollutants in soil, sediment and water. Phytoremediation involves
several processes.
Phytophotolysis
light-mediated degradation
Phytovolatilization
volatilization (release)
to the atmosphere
Phytostabilization
incorporated into plant
structures
Rhizofiltration
adsorption to the roots
Phytoextraction
intake to plant tissues
Rhizodegradation
degradation by
microbes associated
with the root zone
Phytotransformation
transformation by plant enzymes

27. Slota M. 2013 - 27 -
[http://urbanomnibus.net]
POSSIBLE APPLICATIONS
Phytoextraction of heavy metals is recognized as one of the largest economic
opportunity for phytoremediation. Efective phytoextraction requires efficient plants
metal uptake and translocation, ability to sequester them in their tissues and a
high annual production of above-ground biomass.
Two different approaches have been developed:
1) the use of hyperaccumulating plants with exceptional metal accumulating
capacity but usually low biomass;
2) the use of selected conventional high biomass crops usually with the
help of additives to increase heavy metal bioavailability and thus uptake.
(Cosio, 2004)

28. Slota M. 2013 - 28 -POSSIBLE APPLICATIONS
Short-rotation willow coppice (clones of Salix viminalis, S. dasyclados and
S. schwerinii) is cultivated not only to produce biomass for energy, but also to
treat waste products, taking up pollutants from soil and water.
Waste products – mainly urban wastewater, landfill leachate, industrial
wastewaters, sewage sludge and wood-ash – have been successfully applied to
the willow coppice to reduce, through plant uptake, the content of pollutants such
as heavy metals and/or excess nutrients in water and soils.
Industrial waste landfill at Högbytorp in central Sweden covered with short-rotation
willow coppice plantation (A).
(Östman, 2003)
A. B.

29. Slota M. 2013 - 29 -
[http://earthfix.opb.org]
POSSIBLE APPLICATIONS
Starting in 2002, a Texas company called Viridian Resources planted a
selected variety of alyssum (Alyssum corsicum) to recover nickel merging
hyperaccumulating plants for the mining venture.
Company has developed a process known as phytomining, which uses naturally-
occurring plants that have evolved to cope witg nickel-bearing serpentine soils.
Plants accumulates 1.75-2.9% nickel (by weight) in their leaves and can be
burned after harvesting for the nickel recovery.
It is supposed that nickel-rich soils could produce 20 t/ha of nickel-bearing
biomass, which when burned yield an ash at a grade of 30-40% Ni.
Yellow tuft alyssum (Alyssum corsicum ; A) field in Illinios Valley, Oregon (B), which
can be harrwested and burned giving Ni rich ash (C) without the landscape destruction.
A. B. C.

30. Clemens, S. 2010. Zn- a versatile player in plant cell biology. [In]: Hell, R., Mendal, R.R., (eds.)
Cell Biology of Metals and Nutrients, Plant Cell. Monographs 17, Springer Press. Berlin
Heidelberg, s. 281-298
Duffus J. 2002. “Heavy metals" a meaningless term? (IUPAC Technical Report). Pure and
Applied Chemistry 74: 793-807
Gomes, C. D. S. F., & Silva, J. B. P. (2007). Minerals and clay minerals in medical geology.
Applied Clay Science, 36(1), 4-21.
Hall J., 2002. Cellular mechanisms for heavy metal detoxification and tolerance. J. Exp. Bot. 53:
1–11.
Hassinen V.H., Tervahauta A.I., Kärenlampi S.O. 2007. Searching for genes involved in metal
tolerance, uptake, and transport. Methods in Biotechnology 23: 265-289
Östman, M., Wahlberg, O., & Mårtensson, A. (2008). Leachability and metal-binding capacity in
ageing landfill material. Waste management, 28(1), 142-150.
Park H., McGinn P.J., Morel F.M. 2008. Expression of cadmium carbonic anhydrase of diatoms in
seawater. Aquatic Microbial Ecology 51: 183-193
Peer W.A., Mahmoudian M., Freeman J.L., Lahner B., Richards E.L., Reeves R.D. 2006.
Assessment of plants from the Brassicaceae family as genetic models for the study of nickel
and zinc hyperaccumulation. New Phytologist 172: 248-260
Perrier, N., Colin, F., Jaffré, T., Ambrosi, J. P., Rose, J., & Bottero, J. Y. (2004). Nickel speciation
in Sebertia acuminata, a plant growing on a lateritic soil of New Caledonia. Comptes Rendus
Geoscience, 336(6), 567-577.
Prasad M., Strzałka K., 2002. Physiology and biochemistry of metal toxicity and tolerance in
plants. Kluwer Academic Publ., Dordrecht.
Reichman S., 2002. The responses of plants to metal toxicity: a review focusing on copper,
manganese and zinc. Australian Minerals & Energy Environment Foundation, Melbourne.
Slota M. 2013 - 30 -LITERATURE

31. Sagner, S., Kneer, R., Wanner, G., Cosson, J. P., Deus-Neumann, B., & Zenk, M. H. (1998).
Hyperaccumulation, complexation and distribution of nickel in< i> Sebertia acuminata</i>.
Phytochemistry, 47(3), 339-347
Shaw J., 2000. Heavy metal tolerance in plants: evolutionary aspects. CRC Press,
Boca Raton.
Schulze E., Beck E., Müller-Hohenstein K. 2005. Plant ecology. Springer. Berlin, s. 175-196
Talke I.N., Hanikenne M., Krämer U. 2006. Zn-dependent global transcriptional control,
transcriptional de-regulation and higher gene copy number genes in metal homeostasis of the
hyperaccumulator Arabidopsis halleri. Plant Physiology 142: 148-167
Waldron K.J., Rutherford J.C., Ford D., Robinson N.J. 2009. Metalloproteins and metal sensing.
Nature 460: 823-830
Woźny A., A. Goździcka-Józefiak (red.) 2010. Podstawy biologii komórki, tom 2 - Reakcje
komórek roślinnych na czynniki stresowe. Wydawnictwo Naukowe UAM, Poznań pp. 90-150
Wright D., Welbourn P. 2002. Environmental Toxicology. Cambridge University Press. New York,
s. 249-273
Slota M. 2013 - 31 -LITERATURE