Physiological, biochemical and molecular
mechanisms of plant adaptation to heavy metals:
implications for the phytoremediation technology
Massimo Zacchini
Institute of Agro-environmental and Forest Biology (IBAF)
Via Salaria Km 29,300 - 00015 Monterotondo (Rome) Italy
CNR - National Research Council of Italy
Plants have evolved a wide array of basic mechanisms to cope with
the presence of excess metals in the growth substrate
Plant ideotype for phytoremediation of metal
contaminated sites:
High metal tolerance
(detoxification, compartmentalisation, chelation)
High metal accumulation
(uptake, storage, transpiration, biomass)
High metal translocation to aerial parts
(transpiration, metal transporters)
Two approaches for studies on phytoremediation
Hypertolerant – Hyperaccumulator
plants
-Naturally occurring plant species able to
accumulate and safely storing metals in their
tissues up to 10.000 mg kg-1 DW (1%)
-Numerous genera and species, mainly
Brassicaceae (Thlapsi, Alyssum), Asteraceae
(Berckheya), Stackhousiaceae (Stackhousia),
characterised by small biomass and a limited
soil exploration by roots
Limited metal
hectare basis
removal
on
Hypotolerant – Non hyperaccumulator
plants
-Many plant species, herbaceous or woody plants,
characterised by moderate tolerance to metals and
reduced ability to safely accumulate them in their
tissues
-Among them, those able to produce large amount
of biomass and to perform a good soil exploration
(Amaranthus, Zea mays, fast growing woody
species) are good candidates for phytoremediation
Higher metal
hectare basis
removal
on
Salicaceae family
Poplar
Willow
Approx. 400 species distributed worldwide
More than 25 species distributed worldwide
Fast growing (3-5 meters/year), large biomass (10-15 tons DW y-1 hectare-1)
High transpiration rate (100 l/day/5 y-old poplar tree)
Not part of food chain
Industrial uses (paper, bio-energy, furniture etc.)
Long lived (25 to 30 years)
Adaptability to severe soil condition
Grow easily from cuttings
Short Rotation Coppicing (SRC) management
Easy to produce hybrids, high genetic variability
Salicaceae and
phytoremediation
Drawbacks:
Advantages:
Good metal tolerance
Metal accumulation
Large biomass production
Metal translocation to aerial parts
Large soil exploration by roots
Metal dispersion through deciduous foliage
Hydrological control (phytostabilisation)
Ecological restoration
Multiple ecological services (biomass for energy)
Easy association with microorganisms
Our experimental conditions
In vitro culture
Greenhouse
Hydroponic culture
Outdoor system
(Mesocosms)
Metal tolerance and
phytoremediation
Traits of interest
Morpho-anatomical
Metal accumulation pattern
Physiological
Biochemical
Molecular
Morpho-anatomical traits
Poplar plants treated
with 50 µM Cd under
hydroponics
A - Root apex C
B - Root apex Cd
C - Root cross section (Cd)
D - Root hairs (Cd)
Crio-SEM images
Ability of poplar (P. alba clone 14P11) to maintain root integrity
Cocozza et al. (2008) iForest 1:96-103
Morpho-anatomical traits
A – P. alba clone 14P11
B - P. alba clone 6K3
C - P. alba clone 14P11
D - P. alba clone 6K3
Crio-SEM images
No shrinkage or collapse in root cells of Cd-treated plants of both clones
Central cylinder and endodermis showed well preserved cells
Main transport systems not impaired by Cd exposure
Cocozza et al. (2008) iForest 1:96-103
Metal accumulation pattern
EDXMA spectrum of P. alba
roots treated with 50 µM Cd
under hydroponics
Dot map metal distribution
revealed
that
Cd
is
preferentially localised in the
surroundings of the central
cylinder
Cocozza et al. (2008) iForest 1:96-103
Metal accumulation pattern
EDXMA spectrum of P. alba
roots treated with 50 µM Cd
under hydroponics
Cd localisation highlights a
preferential metal accumulation
in the cell walls of the cortex
parenchyma cells
Reduction of metal transport
to aerial plant organs
Cocozza et al. (2008) iForest 1:96-103
Physiological traits
Photosynthesis is strictly related
to transpiration, regulating the
movement of water and solutes
from root to leaves
Photosynthesis and transpiration
are the driving forces to move
metals from growth substrates to
plant organs
Physiological traits
An example of different metal tolerance strategy in poplar and willow
n.d.
Leaf [Cd]
n.d.
22
n.d.
173
P. nigra (Poli), P. x canadensis (A4A) and S. alba (SS5)
plants treated with 50 µM Cd under hydroponics
373
(mg Kg -1)
A- Net photosynthesis
E- Transpiration
Cd-treated willow plants showed the highest metal
concentration and the lowest photosynthesis and
transpiration inhibition
Pietrini et al. (2010) Plant Biology 12:355-363
Physiological traits
Photosynthetic efficiency by CHL fluorescence analysis
Plant - Clone
P. nigra - Poli
[Cd] (mg Kg
22
–1)
Visual symptoms
ΦPSII
NPQ
(Quantum
efficiency of PSII
photochemistry)
(Nonphotochemical
quenching)
(± 5)
P. x canad. - A4A
173 (± 22)
S. alba - SS5
373 (± 25)
In willow plants impairment of the
photosynthesis only in patches
near the main vein
0
1
Physiological traits
Evaluation of Cd presence in necrotic spots by Ed-XRF analysis
Plant - Clone
[Cd] (mg Kg
P. nigra - Poli
22
–1)
(± 5)
P. x canad. - A4A
173 (± 22)
S. alba - SS5
373 (± 25)
In poplar A4A Cd presence in
the whole leaf lamina while in
willow in necrotic spots only
Cd Kα
Metal confinement in areas of “suicide
cells” allows to maintain photosyntetic
efficiency in willow
Pietrini et al. (2010) Plant Biology 12:355-363
Biochemical traits
S-compounds (Thiols, phytochelatins)
Metal binding molecules
O-compounds (Organic acids)
N-compounds (Aminoacids, polyamines)
non–enzymatic (Thiols, polyamines,
phenols, carotenoids)
Antioxidant compounds
enzymatic (APX, CAT, SOD, GPX )
Biochemical traits
Examples of different metal tolerance strategy in poplar and willow
Plant - Clone
P. nigra - Poli
[Cd] (mg Kg
22
–1)
(± 5)
Visual symptoms
PCs HPLC chromatograms
Cd
C
P. x canad. - A4A
173 (± 22)
Cd
C
S. alba - SS5
373 (± 25)
Cd
In willow plants no constitutive or
Cd-induced PCs. The lack of
PCs possibly related to metal
tolerance and accumulation
C
Pietrini et al. (2010) Plant Biology 12:355-363
Biochemical traits
Plant - Clone
[Cd] (mg Kg
–1)
Visual symptoms
Organic acids (µg g –1 FW)
Malic acid
2 100
a
1 950
22
(± 5)
1 800
µ g malic acid / g F.W.
P. nigra - Poli
b
1 650
1 500
c
1 000
c
750
500
250
0
SS5
Willow
Poli
Poplar
S. alba - SS5
Citric acid
373 (± 25)
1200
a
In willow plants metal tolerance and
accumulation associated to both a
constitutively higher OA content and its
Cd-induced increase
µg citric acid / g F.W.
1000
800
b
600
150
c
c
100
50
0
Poli
Poplar
SS5
Willow
Zacchini et al. (2011) Biologia Plantarum 55:383-386
Biochemical traits
Plant - Clone
[Cd] (mg Kg
–1)
Visual symptoms
Polyamines (nmol g –1 FW)
Putrescine
30 0
a
P. nigra - Poli
22
nmol PUT / g F.W.
25 0
(± 5)
a
20 0
15 0
30
b
20
c
10
0
Po li
SS5
Spermidine
35
a
nmol SPD / g F.W .
30
25
20
b
15
b
10
S. alba - SS5
5
373 (± 25)
c
0
P o li
SS5
Spermine
8
a
In willow
plants
metal tolerance
and
n mo l SPM / g F.W .
6
ab
4
b
2
accumulation associated to Cd-induced
n. d
0
Po li
Poplar
S S5
Willow
increase of PA content
Zacchini et al. (2011) Biologia Plantarum 55:383-386
Molecular traits
Example of different metal tolerance ability in two P. nigra clones
P. nigra (Poli) and P. nigra (58-861) plants treated with 50 µM Cd under hydroponics
Gaudet et al. (2011) Tree Physiology 31:1309-1318
Molecular traits
Analysis of gene expression by semi-quantitative RTPCR of some candidate genes for metal tolerance
MT2 – Metallothionein
APX – Ascorbate peroxidase
GR – Glutathione reductase
GSTF- Glutathione S-Transferase
A notable change in in the
expression of GSTF4
Gaudet et al. (2011) Tree Physiology 31:1309-1318
Molecular traits
Quantification of the transcripts
of the gene GSTF4 by real time
PCR and of the thiol content by
HPLC
The content of thiols and PCs
discriminated the biochemical
response of the two genotypes
and
was
consistent
with
expression of the GSTF4 gene
The better tolerance of genotype Poli indicates that the
regulation of GSH-related pathways was effective in
adapting to Cd. The high metal tolerance associated with an
enhanced production of PCs and an increase of GSTF
transcripts, likely occurring to bind Cd to GSH
Poli
Ti 0,91
58-861
Ti 0,42
Gaudet et al. (2011) Tree Physiology 31:1309-1318
Concluding remarks
Metal tolerance and accumulation rely on a complex array of processes
differently characterising plant species
Among these processes, peculiar mechanisms at morpho-anatomical,
physiological and bio-molecular level can be highlighted
To tolerate and accumulate metals, different strategies can be carrried out
by plants, also with similar genetic background
The characterisation of the morpho-physiological and bio-molecular traits
involved in these processes represents a key point in order to assist the
selection of plants for phytoremediation
Early plant genotype screening can be a valid tool to perform this
characterisation
MOBILITY GRANTS TOWARDS INDIA within EU TECO Project
TECO "Technological Eco-Innovations for the Quality Control of Polluted Waters and
Soils" is a project co-funded by the European Union whose goal is to improve and
promote collaborations between Europe and India in the field of environmental
sustainability.
The project provides GRANTS for the mobility of European researchers and experts who
wish to spend a period up to four months in Indian research institutes or private
companies to open new professional collaborations and to develop innovative ideas in
the field of the decontamination of WATERS and SOILS.
The call will stay open in 2016 until the funds are available.
All information can be found on: http://www.tecoproject.eu/index.php/project-submission
Contacts:
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E-Mail: [email protected]
www.tecoproject.eu
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