Evaluation of the phytoremediation potential of Eucalypt hybrid clones
for cadmium by morpho-physiological and biochemical analyses: An
indoor and outdoor study
1Iori
V., 1Pietrini F., 1Bianconi D., 2Mughini G., 1Massacci A., 1*Zacchini M.
Institute of Agro-environment and Forest Biology, National Research Council (CNR), Via Salaria
Km. 29,300, 00015 Monterotondo Scalo (Roma), Italy
2 Research Unit for Intensive Wood Production, The Agricultural Research Council (CRA), Via Valle
della Quistione, 27, 00166 Roma, Italy
1
*Corresponding
author: [email protected]
Among heavy metals, Cd is one of the most hazardous due to its ubiquitous nature, non
biodegradability, high toxicity to biota even at low concentrations and high solubility in water. In this
regard, a great challenge arises from reclaiming municipal wastewater as well as industrial effluents for
safe agricultural use. In recent years, the use of woody plant species in environmental remediation
projects has been increasing. Eucalyptus species is characterised by some interesting traits that indicate
its suitability for soil and water phytoremediation: fast growth, high biomass production, extended root
apparatus, notable tolerance to submersion, adaptability to different climatic and edaphic conditions.
Moreover, eucalypts are suited to plantation of degraded lands, reducing pollutant availability and
leaching and, consequently, increasing soil stabilisation. Little information is available in the literature
on the potential of eucalypt for Cd phytoremediation, in particular for the restoration of contaminated
waters. This work aimed at evaluating the capability of Eucalyptus species to tolerate, accumulate and
translocate Cd in order to assess the potential of this species for the reclamation of polluted waters. To
this end, morpho-physiological and biochemical parameters associated with metal tolerance and
accumulation processes were studied in two hybrid clones of Eucalyptus camaldulensis Dehnh. × E.
globulus subsp. bicostata (Maiden, Blakely & J.Simm.) J.B. Kirkp. (Velino ex 7 and Viglio ex 358)
exposed to 50 μM CdSO4 in hydroponics under greenhouse conditions and in outdoor mesocosms.
About greenhouse experiment, the two eucalypt clones showed a different sensitivity to the metal after
three-week-long treatment, being the tolerance index (Ti) of clone Velino significantly higher than
clone Viglio. Cadmium mostly accumulated in plant roots and, to a lesser extent, in stem, as
highlighted by the low translocation factor (Tf) measured in both clones. Clone Velino showed a higher
bioconcentration factor (BCF) than clone Viglio. Net photosynthesis measurement, chlorophyll
fluorescence images, transpiration values and chlorophyll content revealed a Cd-induced impairment of
physiological processes at the leaf level, which was more evident in clone Viglio. Metal binding and
antioxidative compound content was differentially affected by Cd exposure in the two eucalypt clones.
Particularly, the content of thiols like cysteine and glutathione, organic acids like oxalate and citrate,
and polyamines were markedly modulated in plant organs by metal treatment and highlighted different
defence responses by the clones. These results were confirmed by a nine-week-long experiment in
outdoor mesocosms, filled with agriperlite, showing a good performance of eucalypt in biomass
production and a preferential accumulation of Cd in plant roots, with poor translocation to aerial
organs. This study allowed to highlight an interesting potential of eucalypt plants to realise a double
ecological service by phytoremediating Cd-polluted waters while producing biomass for energy
purpose. Evaluation of the phytoremediation potential of Eucalypt hybrid clones for cadmium by morphophysiological and biochemical analyses: An indoor and outdoor study
Iori V.1, Pietrini F.1, Bianconi D. 1, Mughini G. 2, Massacci A. 1, Zacchini M. 1
1Institute
of Agro-environmental and Forest Biology, National Research Council of Italy (CNR), Via Salaria Km. 29,300, 00015 Monterotondo Scalo (Roma)
E-mail: [email protected]
2Research Unit for Intensive Wood Production, The Agricultural Research Council (CRA), Via Valle della Quistione 27, 00166 Roma
INTRODUCTION
Cadmium (Cd) is one of the most hazardous metals for the environment due to its ubiquitous nature, non biodegradability, high toxicity to biota even at low concentrations and high solubility in water (Xie et al. 2013). In this regard, a great challenge arises
from reclaiming municipal wastewater as well as industrial effluents for safe agricultural use. In recent years, the use of woody plants species in environmental remediation projects has been increasing. Eucalyptus species is characterised by some
interesting traits that indicate its suitability for soil and water phytoremediation: fast growth, high biomass production, extended root apparatus, notable tolerance to submersion, adaptability to different climatic and edaphic conditions (Shah et al. 2011).
Little information is available in the literature on the potential of eucalypt for Cd phytoremediation, in particular for the restoration of contaminated waters. This work aimed at evaluating the capability of Eucalyptus species to tolerate, accumulate and
translocate Cd in order to assess the potential of this species for the reclamation of polluted waters. To this end, morpho-physiological and biochemical parameters associated with metal tolerance and accumulation processes were studied in two hybrid
clones of Eucalyptus camaldulensis Dehnh. × E. globulus subsp. bicostata (Maiden, Blakely & J.Simm.) J.B. Kirkp. exposed to Cd in hydroponics under greenhouse conditions and in outdoor mesocosms.
MATERIALS AND METHODS
Two hybrid clones of Eucalyptus camaldulensis Dehnh. × E. globulus subsp. bicostata (Maiden, Blakely & J.Simm.) J.B. Kirkp. (Velino ex 7 and Viglio ex 358) (Mughini et al. 2012) were exposed to 0 (C) and 50 μM CdSO4 (T) in third-strength Hoagland
solution for three weeks in plastic pots in a greenhouse under natural photoperiod, with mean (night-day) temperatures of 22-25°C and a relative humidity of 60-70%. Air pumping was provided to avoid oxygen deprival. Nutrient solution was replaced twice a
week to maintain initial Cd concentration. Chlorophyll fluorescence imaging analysis was performed on fully expanded leaves by a Imaging-PAM fluorometer (Walz, Effeltrich, Germany). At the end of the treatment, plants were harvested, carefully washed
with distilled water, separated in their organs, sampled for biochemical analyses (Pietrini et al., 2014), dried in a oven at 80°C and finally weighed. Metal content was analysed as elsewhere reported (Zacchini et al. 2009). Parallely, clone Viglio was also
utilised in a nine-week-long experiment in outdoor mesocosms filled with agri-perlite.
400.0
18000
Velino
Viglio
grown in the presence of Cd to dry weight of control plants. The two
a
200.0
b
eucalypt clones showed a different sensitivity to Cd after three-week-long
a
treatment, being the Ti of clone Velino significantly higher than clone Viglio,
0.8
b
0.6
highlighting their high and moderate tolerance to this metal, respectively,
0.4
Remarkable Cd accumulation was detected in the roots, particularly in clone
Velino
Viglio
12000
b
9000
-1
Relative units
a
15000
Cd concentration (mg Kg DW)
300.0
The tolerance index (Ti) is calculated as the ratio of dry weight of plants
Velino, which showed a higher Cd concentration compared to clone Viglio. A
6000
3000
600 _
a
a
notable metal concentration was also found in the stem tissues, while low Cd
6
a
accumulation was detected in the leaves. In both organs, no significant
4
a
difference in Cd accumulation between the eucalypt clones was found.
2
0.2
a
a
according to the scheme proposed by Lux et al. (2004).
0.0
Tf
Ti
0
BCF
Roots
Stem
Leaves
8
Velino
C
T
The bioconcentration factor (BCF) is calculated as the ratio of metal concentration in plant
The photosynthetic efficiency of the two eucalypt clones was affected by Cd treatment. In particular, Cd
organs to metal concentration in the substrate. In this study both eucalypt clones showed a
exposure led to a reduction of the photosynthetic CO2 assimilation rate (A) in both clones, more
relevant potential to bioconcentrate Cd. BCF of clone Velino was notably higher than clone
pronounced in plants of clone Viglio. Metal treatment caused photoinhibition of photosynthesis,
A (µmol CO2 m-2 s-1)
6
4
2
0
characterised by a decrease in the apparent quantum efficiency (Φ) and light-saturated rate of CO2
Viglio. The Cd translocation factor (Tf) is calculated as the ratio of Cd concentration in the aerial
-2
part to Cd concentration in the roots. The data showed a very low Tf in both eucalypt clones,
assimilation (Amax), particularly in clone Viglio. In addition, in the presence of Cd, photorespiration
without any significant difference between them.
process (light compensation point-LCP) increased, especially in clone Viglio, while dark respiration rate
0
200
400
600
800
PPFD (µmol m-2 s-1)
8
(Rd) decreased in both clones.
Viglio
A (µmol CO2 m-2 s-1)
6
Cd treatment caused a decrease in chlorophyll content and
50
an increase in F0 and Fm in both clones. Conversely, in Cd
Total chlorophyll content
4
2
0
Velino
Viglio
40
-2
Chlorophyll content (µg cm )
treated plants a decrease in Fv/Fm was observed in both
clones, more evident in clone Viglio, suggesting a damage
to the PSII centres (Franklin et al. 1992). Finally, the
maintenance of relatively high values of ΦPSII, qP and the
a
-2
a
0
30
200
400
600
800
PPFD (µmol m-2 s-1)
b
c
20
Eucalyptus clones showed no modification of OA content in leaves after Cd
10
exposure, suggesting a lack of involvement of these compounds in the
slight increase of NPQ indicated that Cd treatment affected
0
C
to a lesser extent the photochemical activity in plants of
T
C
different tolerance response. In the roots, Cd treatment caused a remarkable
T
decrease of oxalic acid content in both clones and an enhancement of the
clone Velino compared to clone Viglio.
citric acid content, particularly evident in clone Velino where it can be related
to a higher tolerance to the metal (Zacchini et al. 2011).
content of SPD and SPM in clone
b
8
b
6
Viglio compared to Velino occurred,
c
4
2
c
c
while
the
constitutively more elevated thiol
contents in the leaves of clone
3
related
to
Cd
leaves, except for PUT in leaves.
b
1
b
b
b
6000
80
b
b
60
a
b
ns
40
c
b
20
c
140
SPD in leaves
PUT in roots
c
c
SPM in leaves
a
120
100
a
80
b
b
a
Velino
Viglio
4000
3000
2000
b
c
c
400
a
300
200
a
b
100
b
60
b
b
0
bc
40
c
C
a
T
C
T
C
T
C
T
b
20
c
c
0
Citric acid in roots
Oxalic acid in roots
5000
0
tolerance and accumulation. Cadmium
free PA content both in roots and
a
100
c
treatment of eucalypt plants lowered
2
Velino is consistent with its higher
tolerance to the metal.
inversely
a
-1
Cd
Glutathione in roots
Cysteine in roots
Thiol content (nmol g FW)
to
resulting
0
can be related to its higher
sensitivity
c
a
Velino
Viglio
-1
10
-1
About PAs, a constitutively higher
12
Polyamine content (nmol g FW)
-1
and GSH content in clone Viglio
a
SPM in leaves
-1
level an enhancement of both Cys
Velino
Viglio
a
14
SPD in leaves
PUT in leaves
120
Polyamine content (nmol g FW)
leaves in the two clones: at root
Thiol content (nmol g FW)
induction of thiols in the roots and
Glutathione in leaves
Cysteine in leaves
16
Organic acid content (µg g FW)
Cd treatment caused a differential
0
C
T
C
T
C
T
C
T
C
T
C
T
C
T
C
T
C
T
C
T
200.0
experiment
of
in
the
nine-week-long
outdoor
mesocosms,
confirmed those obtained in hydroponics
highlighting
a
good
performance
of
eucalypt in biomass production and a
preferential accumulation of Cd in plant
Viglio
-1
results
Cd concentration (mg Kg DW)
The
180.0
a
160.0
140.0
2.0
1.5
b
1.0
0.5
0.0
roots, with poor translocation to aerial
Roots
Aerial part
organs.
CONCLUSIONS
REFERENCES
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The results of this work show the ability of eucalypt plants to notably bioconcentrate Cd in plant roots and, to a lesser extent, in stem. The
confinement of the metal accumulation in the roots, observed in both Eucalyptus clones by the high root BCF and the low Tf values, can avoid metal
to accumulate in the leaves and then strongly affect leaf functionality. Nevertheless, for phytoremediation purposes, it has been pointed out that high
metal accumulation in roots should not be considered a drawback (Iori et al., 2013). In fact, remediation processes of polluted waters in wetlands,
mesocosms, and other technological systems also allow harvesting of the below-ground biomass at the end of the decontamination cycle, thus
removing the metal accumulated by the root system. These findings, together with the relevant tolerance to submersion of eucalypt species, open
interesting perspectives for the utilisation of this plant species for remediation of wastewater in water-based decontamination systems.