Copper-Induced Responses in Poplar Clones are Associated with Genotype- and Organ-Specific Changes in Peroxidase Activity and Proline, Polyamine, ABA, and IAA Levels

Abstract

The involvement of auxin, abscisic acid (ABA), polyamines (PAs), and proline in adaptation to long-term exposure of woody plants to high levels of heavy metals in soil has received scant attention, even in poplar which is a good candidate for phytoremediation of metal-polluted soils and is regarded as a model for basic research in tree species. Three poplar clones (M1, PE19/66, and B229) were comparatively analyzed in a pot experiment for their responses to 300 mg kg⁻¹ Cu(NO3)2 at morphological, physiological, and biochemical levels. After 4 months, despite the prevalent accumulation of Cu in roots, where the metal reached potentially toxic concentrations, the three clones showed distinct Cu accumulation and translocation capacities, whereas they did not display evident toxicity symptoms or growth inhibition. Several protective mechanisms, namely decreased photosynthetic functionality, enhanced guaiacol peroxidase (GPOD) activity, and accumulation of proline and PAs, were differentially activated in Cu-treated plants in an organ- and clone-specific manner. Overall, a positive relationship between root Cu concentration with GPOD, proline, and PAs was observed. In M1, higher Cu accumulation in roots and leaves compared with other clones was reflected in stimulation of GPOD activity in both organs and in enhanced proline, and PA levels. In PE19/66, these responses were observed only in roots concomitant with high Cu accumulation. Clone B229 accumulated very low amounts of Cu, therefore, these defense responses were attenuated compared with other clones. Enhanced ABA concentrations in response to Cu were observed in PE19/66 and B229; this was likely responsible for stomatal limitation of photosynthesis in PE19/66, whereas in B229 this effect may have been counteracted by increased IAA. Essentially unchanged leaf auxin levels under Cu stress may account for the lack of shoot growth inhibition observed in all three clones; B229 was the only clone that displayed Cu-induced IAA accumulation in roots. Results are discussed in terms of clone-specific adaptive mechanisms to Cu stress in tolerant poplars.

Full text

Copper-Induced Responses in Poplar Clones are Associated
with Genotype- and Organ-Specific Changes in Peroxidase
Activity and Proline, Polyamine, ABA, and IAA Levels
Marko Kebert
1
•Francesca Rapparini
2
•Luisa Neri
2
•Gianpaolo Bertazza
2
•
Sas
ˇa Orlovic
´
1
•Stefania Biondi
3
Received: 5 April 2016 / Accepted: 30 June 2016
ÓSpringer Science+Business Media New York 2016
Abstract The involvement of auxin, abscisic acid (ABA),
polyamines (PAs), and proline in adaptation to long-term
exposure of woody plants to high levels of heavy metals in
soil has received scant attention, even in poplar which is a
good candidate for phytoremediation of metal-polluted
soils and is regarded as a model for basic research in tree
species. Three poplar clones (M1, PE19/66, and B229)
were comparatively analyzed in a pot experiment for their
responses to 300 mg kg
-1
Cu(NO
3
)
2
at morphological,
physiological, and biochemical levels. After 4 months,
despite the prevalent accumulation of Cu in roots, where
the metal reached potentially toxic concentrations, the
three clones showed distinct Cu accumulation and
translocation capacities, whereas they did not display evi-
dent toxicity symptoms or growth inhibition. Several pro-
tective mechanisms, namely decreased photosynthetic
functionality, enhanced guaiacol peroxidase (GPOD)
activity, and accumulation of proline and PAs, were dif-
ferentially activated in Cu-treated plants in an organ- and
clone-specific manner. Overall, a positive relationship
between root Cu concentration with GPOD, proline, and
PAs was observed. In M1, higher Cu accumulation in roots
and leaves compared with other clones was reflected in
stimulation of GPOD activity in both organs and in
enhanced proline, and PA levels. In PE19/66, these
responses were observed only in roots concomitant with
high Cu accumulation. Clone B229 accumulated very low
amounts of Cu, therefore, these defense responses were
attenuated compared with other clones. Enhanced ABA
concentrations in response to Cu were observed in PE19/66
and B229; this was likely responsible for stomatal limita-
tion of photosynthesis in PE19/66, whereas in B229 this
effect may have been counteracted by increased IAA.
Essentially unchanged leaf auxin levels under Cu stress
may account for the lack of shoot growth inhibition
observed in all three clones; B229 was the only clone that
displayed Cu-induced IAA accumulation in roots. Results
are discussed in terms of clone-specific adaptive mecha-
nisms to Cu stress in tolerant poplars.
Keywords Abscisic acid Auxin Heavy metals 
Phytoremediation Polyamines Populus
Introduction
Copper (Cu), an important redox component and enzyme
cofactor, is an essential trace element with several func-
tions in plant metabolism, namely photosynthesis, hormone
signaling, and mitochondrial respiration (Yruela 2009) and,
hence, required for normal growth and development.
However, the redox properties that make Cu an essential
element also contribute to its inherent toxicity (Cuypers
and others 2011). In the cell, Cu(II) is easily reduced to the
unstable Cu(I) which, by giving rise to Fenton-type reac-
tions, can generate reactive oxygen species (ROS) and,
consequently, may cause oxidative stress (Cuypers and
others 2009). In addition, Cu can displace other divalent
&Francesca Rapparini
f.rapparini@ibimet.cnr.it
1
Institute of Lowland Forestry and Environment (ILFE),
University of Novi Sad, A. C
ˇehova 13, 21000, Novi Sad,
Serbia
2
Institute of Biometeorology, National Research Council,
(IBIMET-CNR), via P. Gobetti 101, 40129 Bologna, Italy
3
Department of Biological, Geological and Environmental
Sciences (BiGeA), University of Bologna, via Irnerio 42,
40126 Bologna, Italy
123
J Plant Growth Regul
DOI 10.1007/s00344-016-9626-x

cations coordinated with macromolecules, causing their
inactivation or malfunction (Murphy and others 1999).
Thus, Cu is phytotoxic at high concentrations (Xu and
others 2006).
Industrial (e.g., smelting) and agricultural (e.g., fertil-
izers used in intensive farming) activities can lead to
contamination of soils and ground water with potentially
toxic amounts of Cu (Borghi and others 2007). Phytore-
mediation may represent an environmentally friendly way
of reclaiming heavy metal (HM)-polluted soils (Pulford
and Watson 2003) and fast-growing trees, such as willows
and poplars, have been indicated as good candidates for
this purpose (Dos Santos Utmazian and others 2007).
In accordance with the high genetic variability in the
Salicaceae family (Smulders and others 2008), most studies
reveal that tolerance, metal uptake capacity, and organ
allocation are highly variable among Populus species (He
and others 2013), even at the intraspecific (clonal) level
(Laureysens and others 2004; Dos Santos Utmazian and
others 2007; Borghi and others 2007; Castiglione and
others 2009; Gaudet and others 2011). The accumulation
capacity and growth responses of poplars after short-term
or long-term exposure to high concentrations of HMs, such
as Cu, cadmium (Cd) and zinc, have been investigated in
numerous genotypes and under different experimental
conditions (in vitro, hydroponic or pot experiments, and
field trials). Recent developments in the functional geno-
mics, molecular genetics, and biology of Populus reinforce
this plant as the model tree for studies on the physiological
and molecular mechanisms of tolerance to abiotic stress
(Wullschleger and others 2009) over the standard small-
sized Arabidopsis thaliana (Marmiroli and others 2013)
commonly used to investigate plant responses to Cu
(Table 1). Comparing different genotypes may also shed
further light on the mechanisms for HM detoxification and
tolerance in poplar, and allow better exploitation of poplar
clones for phytoremediation purposes.
Exposure to HMs can lead to activation of enzymatic
defense mechanisms and to accumulation of other protec-
tive metabolites (nonenzymatic antioxidants), such as the
amino acid-derivatives polyamines (PAs) and proline, for
counteracting metal-induced oxidative stress. An analysis
of the root transcriptome of a Cu-tolerant P. deltoides clone
revealed that most of the transcripts upregulated by Cu
were related to defense against antioxidant mechanisms,
metal homeostasis, and water flux control (Guerra and
others 2009). The activity of the antioxidant enzyme
superoxide dismutase was reported to change in response to
nickel, Cd, and Cu in different Populus genotypes (Trudic
´
and others 2012). Peroxidases also play a key role against
oxidative stress and the accumulation of ROS in response
to HM stress (Kim and others 2010). These enzymes are
also involved in lignification, suberization, and crosslink-
ing of cell wall compounds.
PAs modulate plant tolerance to HM stress. They may
act as protectants, by directly scavenging ROS (Velikova
and others 2007) or by activating the antioxidant machin-
ery leading to reduced oxidative damage (Velikova and
others 2000; Mandal and others 2013) and may act as
signal molecules in the ABA- and H
2
O
2
-regulated stress
response pathways (Minocha and others 2014). In several
studies, increased levels of the most abundant PAs in
Table 1 Effects of exogenous Cu supplied in the growth medium on endogenous content (EC) and expression of genes (GE) involved in ABA or
IAA synthesis, conjugation, and transport in different species and plant parts
Plant species Type of culture [CuSO
4
] DAT Plant part ABA IAA References
GE EC GE EC
Pisum sativum Hydroponic 0–100 lM 4 Root ?Chaoui and El Ferjani (2005)
Raphanus sativus L. Hydroponic 0.2 mM 7 Whole plant ?– Choudhary and others (2010)
A. thaliana Hydroponic 0–75 lM 14 Root ?
a
Lequeux and others (2010)
A. thaliana In vitro 0–50 lM 7 Shoot, root ?
b
?
a,b
Pet}
o and others (2011)
Raphanus sativus L. In vitro 0.2 mM 7 Whole plant ??– – Choudhary and others (2012)
A. thaliana In vitro 0–60 lM\1 Root ??
a
Yuan and others (2013)
Atriplex halimus Hydroponic 400 lM 30 Leaf, root – ?
b
Bankaji and others (2014)
Suaeda fruticosa Hydroponic 400 lM 30 Leaf, root -
b
?
b
Bankaji and others (2014)
Cucumis sativus Hydroponic 2 mM 3 Seed – ?Wang and others (2014)
A. thaliana In vitro 20–40 lM 8–12 Shoot, root ??
a,b
Wang and others (2015)
a
IAA content measured by DR5::GUS
b
Changes detected only in roots, though analyzed also in shoots
DAT numbers of days after treatment
J Plant Growth Regul
123

plants, putrescine (Put), spermidine (Spd), and spermine
(Spm), in the free and/or conjugated forms, have been
positively correlated with improved HM tolerance
(Bhardwaj and others 2014). Cu-induced changes in
endogenous levels of PAs have been reported in herba-
ceous species (Table 1) as well as in poplar (Franchin and
others 2007; Lingua and others 2008; Castiglione and
others 2009) where enhanced spermidine synthase gene
expression associated with improved growth in mycor-
rhizal poplars grown in the presence of Cu- and Zn-con-
taminated soil suggested that PAs may have a protective
role with respect to HM stress (Cicatelli and others 2010).
Although proline has long been considered a compatible
osmolyte mainly involved in salt and drought stress, recent
results highlight its multiple functions in stress signaling
and adaptation. Szabados and Savoure
´(2010) reviewed the
role of proline in cellular homeostasis, including redox
balance, and suggested that proline modulates gene
expression, possibly essential for plant recovery from
stress. Proline accumulation in plants is mediated by both
abscisic acid (ABA)-dependent and -independent signaling
pathways (Ashraf and Foolad 2007 and references therein).
Increased proline levels have been reported in radish
seedlings exposed to Cu and Cr(VI); mitigation of HM
stress by exogenous epibrassinolide was associated with
higher proline levels as compared with seedlings grown
under HMs alone (Choudhary and others 2010,2011).
Plants also respond to excessive metal exposure with a
fine tuning of endogenous pools of plant hormones, such as
auxin (e.g., indole-3-acetic acid, IAA) and ABA (Elobeid
and Polle 2012; Gangwar and others 2014; Wang and
others 2015), in order to maintain physiological levels
optimal for normal growth and functioning of physiologi-
cal processes. Although the involvement of IAA and ABA
has been established in many abiotic stress responses (Song
and others 2014;Do
¨rffling 2015; Rademacher 2015) and
their exogenous application appears to alleviate the nega-
tive effects of excess HMs on plant growth (Elobeid and
Polle 2012; Ashger and others 2015), investigations on
their endogenous levels after HM exposure are scarce
(Table 1). Both plant hormones are directly involved in
growth responses of the primary sensing organ (i.e., the
root) to excess metals, including Cu (Vitti and others 2014;
Wang and others 2015). However, depending on the organ,
IAA and ABA may have synergistic or opposite effects on
plant growth and development under stress conditions
(Popko and others 2010). In several plant species grown
under HMs, such as Cu, IAA, and ABA were reported to
influence plant growth, but also to regulate oxidative stress
and the antioxidant defense system (Gangwar and Singh
2011; Chaoui and El Ferjani 2005). Concomitant changes
in IAA, ABA, and PA concentrations in response to Cu,
coupled with enhanced antioxidant potential and tolerance
in Raphanus sativus seedlings treated with exogenous plant
growth regulators reveal complex interactions (Choudhary
and others 2010,2012; Yusuf and others 2016). Under-
standing the interplay between protective components of
the antioxidant defense system and plant hormones with a
physiological role in enhancing metal tolerance may con-
tribute to developing physiological, biochemical, and
biotechnological strategies of tolerance against this abiotic
stress. However, very little information is available
regarding plant hormones in HM stress in woody species,
how they are modulated in the different plant parts, and
whether they are genotype-specific. It has been suggested
that differences in sensitivity of poplar genotypes to HM
stress may involve modulation of plant hormone levels and
distribution (Popko and others 2010; Tognetti and others
2012 and references therein).
The overall aim of this work was to provide information
regarding the involvement of the plant hormones IAA and
ABA, and of PAs and proline in poplar adaptation to long-
term exposure to excess Cu in soil. Knowing that there are
strong genotype-dependent responses to HMs in this genus,
we comparatively analyzed three different clones. After
comparatively evaluating the response of the poplar clones
to a high concentration of soil Cu in terms of growth, and
photosynthetic functionality, we addressed the following
questions: (i) are protective/antioxidant responses (peroxi-
dase, PAs, proline) induced by Cu? (ii) Do ABA and IAA
levels change in response to Cu? (iii) Do all of these
changes correlate with the amount of Cu accumulated by
the plant? (iv) Are there clone-specific adaptive responses
to Cu?
This is the first study to consider these aspects in a
woody species after long-term exposure to Cu, and the
results may offer new insight for better exploitation of the
phytoremediation capacity of poplar genotypes.
Materials and Methods
Plant Material and Experimental Design
Cuttings of three poplar clones, M1 (Populus x eurameri-
cana (Dode) Guinier; Hungary), PE19/66 (P. deltoides
Bartr. ex Marsh.; Italy), and B229 (P. deltoides; Serbia),
were planted in spring (early May) in 10-L pots containing
sandy fluvisol soil characteristics of which are reported in
Trudic
´and others (2012). These clones were already
reported as showing differential responses to Cu (Trudic
´
and others 2012). Pots were kept in a greenhouse in
semicontrolled conditions at the Dept. of Biology and
Ecology, Faculty of Science, University of Novi Sad
(Serbia). Soil was previously (end of February) artificially
contaminated by adding Cu(NO
3
)
2
to a final concentration
J Plant Growth Regul
123

of 300 mg kg
-1
soil, corresponding to three times the
Maximum Allowed Amount according to Serbian legisla-
tion (Official Bulletin of the Republic of Serbia No. 52/
2002). Control soil was not artificially contaminated.
Before planting nonrooted cuttings, the soil was left for
8 weeks in order to stabilize metal content through
microflora activity. Each pot contained four cuttings, and
each treatment had three replicates (12 plants per treatment
in total). Pots were watered regularly and Hoagland’s
solution was added once a month. The experiment was
ended after 4 months of Cu treatment, at which time plant
growth was assessed in terms of stem height and diameter.
At the end of the experiment, after gas exchange mea-
surements were performed, fully developed leaves were
harvested, and roots were separated from the substrate by
careful washing under running tap water. One part of the
plant material was oven-dried at 70 °C for 24 h, whereas
the other was frozen in liquid nitrogen. Frozen plant
material was lyophilized for plant hormone (polyamines,
proline, IAA, ABA) analyses. Oven-dried material was
used for Cu determination, and fresh leaves were used for
pigment determination, and guaiacol peroxidase activity.
Metal Content Determination
Approximately 0.3 g each of oven-dried leaves and roots
were ground and homogenized in a laboratory mill and then
digested in 10 mL nitric acid and 2 mL 30 % (v/v) hydrogen
peroxide using a microwave-assisted digestion system (D
series; Milestone, Bergamo, Italy) for 45 min at 180 °C
using a microwave power of 900 W. Homogenates were then
diluted to 25 mL with deionized water. Pretreated samples
were processed in Atomic Absorption Spectrophotometer
(model FS AAS240/GTA120, Varian) using the acetylene/
air burner flame technique for Cu and magnesium quantifi-
cation, whereas a nitrous oxide–acetylene flame was used for
calcium content determination. By means of single-element
hollow-cathode lamps, concentrations of Cu, magnesium,
and calcium were determined at 324.8, 285.2, and 422.7 nm,
respectively, and expressed in mg/kg dry weight (DW) of
plant material. The Translocation Factor (TF) for Cu was
calculated as the ratio between Cu concentration in leaves
and in roots. Phosphorus concentrations were determined
spectrophotometrically using molybdenum blue (Pulliainen
and Wallin 1994).
Gas-Exchanges Measurements
Photosynthetic measurements were performed with a
portable infra-red based gas exchange analyzer (LCpro?;
ADC, BioScientific Ltd) at saturating light intensity
(1000 lmol m
-2
s
-1
). A constant gas flow into the leaf
chamber was measured under ambient humidity and
temperature and monitored by a thermocouple placed
under the leaf. Net photosynthesis (A; lmol CO
2
m
-2
s
-1
),
stomatal conductance (gs; mmol H
2
Om
-2
s
-1
), and
evapotranspiration (E; mmol H
2
Om
-2
s
-1
) were calcu-
lated according to von Caemmerer and Farquhar (1981).
Chlorophyll Determination
Chlorophyll a(chla) and chlorophyll b(chlb) concentra-
tions were measured and calculated according to Lichten-
thaler and Wellburn (1983). About 0.2 g fresh weight (FW)
of leaves or roots was homogenized with 10 mL of 80 %
acetone. The extract was centrifuged at 3000 g for 5 min.
The upper phase was transferred into a quartz cuvette and
its absorbance rates measured at 662 and 644 nm for
chlaand chlb,, respectively, with acetone 80 % as a blank.
Guaiacol Peroxidase Activity Assay
Guaiacol peroxidase (GPOD, EC 1.11.1.7) activity was
assayed according to Zimmerlin and others (1994) with
slight modifications. Activity was determined in 0.1 M
acetate buffer (pH 7.0), in the presence of 0.1 mM H
2
O
2
and 10 mM guaiacol as substrates. Activity was measured
as the increase of absorbance at 436 nm during 2 min. The
extinction coefficient for tetraguaiacol (e=25.6 mM
-1
cm
-1
) was used to calculate enzyme activity, expressed as
enzyme units (U) g
-1
FW.
Proline Analysis
Proline concentration was estimated following the method of
Bates and others (1973) with slight modifications. About
40 mg of freeze-dried tissue (leaves and roots) was crushed
in 1.5 mL of 3 % 5-sulphosalicylic acid, and the homogenate
centrifuged at 16,000 gat room temperature for 20 min. A
1.0 mL aliquot of the supernatant was mixed with 1 mL
ninhydrin reagent [2.5 % ninhydrin in glacial acetic acid-
distilled water-85 % orthophos phoric acid (6:3:1)] and 1 mL
acetic acid. The reaction mixtures were kept in a water bath at
90 °C for 1 h to develop the color. Test tubes were then
cooled in an ice-bath, and 3 mL toluene added to separate the
chromophore. Absorbance of the toluene phase was read in a
spectrophotometer at 520 nm, and proline concentration
calculated according to the previously constructed calibra-
tion curve in a range of 1–500 lmol standard proline.
HPLC Analysis of Polyamines
Plant tissues (approx. 20 mg DW of freeze-dried material)
were extracted with 2 mL 4 % perchloric acid (PCA); the
homogenate was kept for 1 h on ice and then centrifuged at
15,0009gfor 30 min. Aliquots of the supernatants and
J Plant Growth Regul
123

standard solutions of putrescine (Put), spermidine (Spd),
and spermine (Spm) were derivatized with dansyl chloride
as described by Scaramagli and others (1999). Dansylated
derivatives were extracted with toluene, taken to dryness
and resuspended in acetonitrile prior to HPLC analysis.
Aliquots of the supernatant were subjected to acid
hydrolysis (6 M HCl overnight at 110 °C) in order to
release PAs from their PCA-soluble conjugates; released
PAs were derivatized and analyzed as described above.
PAs were separated and quantified by HPLC (Jasco,
Tokyo, Japan) using a reverse phase C
18
column (Spher-
isorb ODS2, 5-lm particle diameter, 4.6 9250 mm,
Waters, Wexford, Ireland), and a programmed acetonitrile–
water step gradient, as previously described (Scaramagli
and others 1999). Eluted peaks were detected by a spec-
trofluorometer (ex. 365 nm, em. 510 nm); the internal
standard used was heptamethylenediamine. Soluble-con-
jugated PA contents were calculated by subtracting the free
PAs in the nonhydrolyzed supernatant from the PAs in the
hydrolyzed supernatant.
Determination of IAA and ABA
Approximately 0.1–0.2 g DW of freeze-dried leaves and
roots were extracted with a mixture (65:35, v/v) of iso-
propanol and 0.2 M imidazole buffer, pH 7.0 to which
[
13
C
6
]IAA and [
2
H
4
]ABA were added as internal standards
for quantitative mass-spectral analysis. After overnight
isotope equilibration, the extraction and purification were
performed according to Chen and others (1988) for IAA
and to Baraldi and others (1995) for ABA. Zones of elution
corresponding to hormonal standards, IAA and ABA, were
collected, evaporated, and treated with diazomethane and
analyzed through capillary gas chromatography-mass
spectrometry-single ion monitoring (GC–MS-SIM) were
performed according to Baraldi and others (1995).
Statistical Analysis
Three types of statistical analyses were carried out for all
dependent variables measured in the experiment. Signifi-
cant differences (i) between controls and Cu treatment
within a clone, and (ii) between clones (controls and Cu-
treated) were tested by Tukey’s honestly significant dif-
ference (HSD) post hoc test for multiple comparisons when
the one-way analysis of variance (ANOVA) indicated
significant differences (P\0.05). Differences were con-
sidered significant at P\0.05. To determine the overall
significances between groups, a two-way factorial analysis
of variance (two-way ANOVA) was used with Cu treat-
ment and clone as factors. Unless specified, the levels of
significance indicated in the text are referred to the two-
way ANOVA for clone and treatment and the interaction.
For the gas-exchange parameters, measurements were
made on six separate leaves; for analytic parameters three
separate extractions were made. Regression analysis was
used to detect the influence of endogenous Cu content on
proline, PA, ABA, and IAA concentrations at the leaf, root,
or whole plant level, determining if the parameters of the
regression are significantly different from zero. All statis-
tical analyses were carried out with SAS 9.4 (SAS Institute,
Cary NC, USA).
Results
Genotype-Dependent Cu Accumulation and Organ
Allocation
Soil Cu concentrations ranged from about 27 to 360–
377 mg kg
-1
DW in control and Cu-supplemented soil,
respectively, at the end of the experiment without significant
differences between clones (data not shown). Under control
conditions, the three clones exhibited similar leaf (ca.
8–12 mg kg
-1
DW) and root (ca. 60–80 mg kg
-1
DW) Cu
concentrations (Fig. 1). After Cu treatment, poplar clones
showed an increase in Cu concentrations in both organs.
Most of the metal was accumulated in the roots and only a
small amount was translocated to the leaves (Fig. 1a, b).
Indeed, the leaf-to-root ratio, or TF, ranged from 0.14 to 0.22
on unsupplemented soil and declined to 0.02–0.05 after
exposure to Cu (data not shown). Cu accumulation in leaves
was dependent on the clone: it increased twofold relative to
control levels in clone M1 and slightly, but not significantly,
in B229 and PE19/66 (Fig. 1a; clone*treatment P\
0.0001). In all three clones, this increase was much smaller
than the one observed in roots where accumulation also
varied depending on the clone (Fig. 1b; clone*treatment
P\0.0001). Thus, Cu treatment induced an almost tenfold
increase in root Cu concentrations relative to controls in
clones M1 and PE19/66, reaching final concentrations of
731 ±16 and 591 ±28 mg kg
-1
DW, respectively; clone
B229 accumulated a significantly lower amount of Cu
(221 ±22 mg kg
-1
DW) compared with the other clones.
Stem Cu concentrations in Cu-exposed plants ranged from
ca. 9 to 13 mg kg
-1
DW (data not shown).
Cu treatment did not substantially affect leaf and root
calcium, magnesium, and phosphorus concentrations rela-
tive to untreated controls in any of the three clones (data
not shown).
Effects of Cu treatment on Photosynthetic
Functionality and Chlorophyll Content
Under control conditions, net photosynthetic (A) and
stomatal conductance (g
s
) rates ranged from about 11.3 to
J Plant Growth Regul
123

14.8 lmol m
-2
s
-1
and 0.2 to 0.5 mmol m
-2
s
-1
,
respectively (Table 2). In each of the three clones, A, g
s
,
and evapotranspiration rate (E) were downregulated by Cu
treatment relative to control values; the extent of the
reduction induced by Cu depended on the clone (Table 2).
In clones M1 and PE19/66 grown on Cu, rates of A were
ca. 50 %, g
s
40 %, and E 60 % of control levels, whereas
in clone B229, reductions were slightly less dramatic.
Cu treatment slightly but significantly decreased
chlaand chlbconcentrations in all clones with genotype-
dependent differences; in PE19/66, the decline was much
stronger than in M1 and B229 (Table 2). Thus, total
chlorophyll levels declined most dramatically (nearly
fivefold) in PE19/66 as compared with controls and only by
about 15–25 % in the other clones.
Guaiacol Peroxidase Activity in Response to Excess
Cu
GPOD activity increased significantly (ca. twofold) in leaves
of M1 and B229 after Cu treatment relative to controls, but not
in PE19/66, whereas in roots, activity increased (threefold)
also in PE19/66 and to a lesser extent (twofold) in M1
(Fig. 2a). Consequently, M1 exhibited an increase in activity
in both organs, while in PE19/66 it increased only in roots, but
not in leaves. Plotting the logarithm of enzyme activity against
the reciprocal of organ Cu concentration showed that enzyme
activity increased with increasing Cu content in both leaves
and roots (Fig. 3). This relationship has been applied to cal-
culate the threshold values for phytotoxicity of metals defined
as the concentration in the plant tissue above which growth is
Fig. 1 Copper concentrations
in leaves (a) and roots (b)of
poplar clones M1, B229, and
PE19/66 after 4 months of
growth in pots containing soil
supplemented (Cu) or not
(Control) with 300 mM CuNO
3
.
Asterisks (*P\0.05,
**P\0.01, ***P\0.001)
indicate significant differences
within each clone between
control and Cu-treated plants
using Tukey’s HSD post hoc
test. Data represent the
mean ±SE (n=3–5)
J Plant Growth Regul
123

reduced in the plant tissue or metabolism changes (Van
Assche and Clijsters 1990; Mocquot and others 1996). In the
examined poplar clones, GPOD activity was stimulated when
Cu contents exceeded about 15–20 mg kg
-1
DW and ca.
250 mg kg
-1
DW in leaves and roots, respectively.
Cu-Induced Accumulation of Proline
Proline concentration ranged from 0.4 to 2.5 mmol g
-1
DW
in all clones under control conditions and Cu treatment.
Upon Cu treatment an increase (ca. 60 %) in leaf proline
level occurred only in PE19/66, whereas in root s a strong Cu-
induced proline accumulation was observed in M1 as well as
in PE19/66, with almost five- and fourfold increases,
respectively (Fig. 2b). Root proline showed a strong linear
relationship with endogenous Cu accumulated after expo-
sure to the metal (r
2
=0.79 P\0.05; data not shown).
Changes in Leaf and Root Concentrations of Free
and Conjugated PAs
Within the free PA pool, Put was the most abundant poly-
amine (ca. 80–180 and 90–150 nmol g
-1
DW in leaves and
roots, respectively, of control plants), followed by Spd and
Spm (Fig. 4a–c) in all three clones. After exposure to Cu,
clones M1 and B229 exhibited the most significant increases
in free PAs in leaves and/or roots, but the responses varied
depending on the clone, the organ, and the individual PA.
Thus, free Put increased about twofold relative to controls in
leaves of M1 and B229, but not in clone PE19/66 (Fig. 4a).
Different from Put, Cu treatment did not generally affect free
Spd and Spm levels in leaves, independently of the clone
(Fig. 4b, c). Similar to leaves, free Put also accumulated
significantly (ca. 80 % more than in controls) in roots of Cu-
treated plants of M1 and B229, but to a lesser extent in PE19/
66 (Fig. 4a). In roots, free Spd levels increased markedly in
response to Cu in clones M1 and B229 (ca. four and twofold,
respectively, compared with controls), but remained unal-
tered in PE19/66 (Fig. 4b; clone*treatment P\0.0001).
Free Spm, like Spd, increased dramatically in roots of clone
M1 exposed to Cu, while it was slightly or not at all affected
by metal treatment in the other two clones (Fig. 4c;
clone*treatment P\0.01).
Put was also the most abundant PA in the conjugated
fraction (ca. 150–325 nmol g
-1
DW in leaves and ca.
130–270 nmol g
-1
DW in roots of control plants, Fig. 4d).
Total conjugated PAs increased markedly after Cu treatment
in both leaves and roots of M1 and B229, with a two to
threefold increase of conjugated Put; in the latter clone, there
was a more pronounced increase at the leaf than at the root
level. By contrast, in PE19/66 conjugated Put did not
increase in leaves but only in roots (Fig. 4d). Conjugated
Spd remained essentially unchanged in leaves of Cu-treated
Table 2 Net photosynthesis (A; lmol m
-2
s
-1
), stomatal conductance (g
s
; mmol m
-2
s
-1
), evapotranspiration rate (E; mmol m
-2
s
-1
), and chlorophyll concentrations (Chla, Chlb,
Chla?Chlb;mgg
-1
FW) in poplar clones M1, B229, and PE19/66, after 4 months growth in pots containing soil supplemented (Cu) or not (Control) with 300 mM Cu(NO
3
)
2
Parameter M1 B229 PE19/66 Two-way ANOVA
Control Cu Control Cu Control Cu Clone Cu Clone 9Cu
A 14.79 ±0.05
a
7.07 ±0.23
b
11.03 ±0.11
a
6.88 ±0.10
b
11.28 ±0.75
a
5.49 ±1.32
b
\0.0001 \0.0001 \0.0001
g
s
0.45 ±0.01
a
0.18 ±0.02
b
0.25 ±0.00
a
0.11 ±0.01
b
0.17 ±0.02
a
0.07 ±0.01
b
\0.01 \0.0001 \0.01
E 2.39 ±0.01
a
1.34 ±0.12
a
2.84 ±0.02
a
2.06 ±0.16
b
2.23 ±0.13
a
1.39 ±0.21
b
\0.0001 \0.0001 \0.05
Chla4.04 ±0.80
a
3.46 ±0.33
b
1.76 ±0.01
a
1.34 ±0.18
a
3.93 ±0.52
a
0.72 ±0.02
b
\0.0001 \0.0001 \0.0001
Chlb1.00 ±0.26
a
0.87 ±0.07
b
0.49 ±0.02
a
0.36 ±0.05
a
1.16 ±0.16
a
0.20 ±0.04
b
\0.0001 \0.0001 \0.0001
Chla?Chlb5.04 ±0.62
a
4.33 ±0.40
b
2.25 ±0.03
a
1.67 ±0.23
a
5.13 ±0.43
a
0.92 ±0.06
b
\0.0001 \0.0001 \0.0001
Different letters indicate statistically significant (P\0.05) differences within clones using Tukey’s HSD post hoc test. Values are mean ±SE of n=3–6
Pvalues of analysis of two-way variance (two-way ANOVA) to test for the effects of clone, Cu treatment (Cu), clone 9Cu treatment, on the different parameters are shown
J Plant Growth Regul
123

plants, but accumulated strongly in roots of all three clones,
again especially in M1 (Fig. 4e). Finally, conjugated Spm,
like the free form, increased significantly under Cu treatment
only in roots of clone M1 (Fig. 4f). The total content of free
and conjugated PAs was positively and significantly corre-
lated with Cu concentration in both leaves (r
2
=0.82
P\0.02) and roots (r
2
=0.75 P\0.03).
Leaf and Root ABA and IAA Levels in Cu-Treated
Plants
In control plants, ABA concentration ranged from 0.38 to
0.65 nmol g
-1
DW in leaves and from 0.44 to 0.84 nmol g
-1
DW in roots (Fig. 5a). Leaf ABA concentration was signifi-
cantly enhanced after Cu treatment in clone B229 (ca. twofold)
and, especially, in PE19/66 (almost threefold), but not in M1; a
similar trend was observed in roots where the only significant
increase (approx. 70 %) relative to control levels occurred in
clone PE19/66 (Fig. 5a).
IAA concentrations were higher in roots
(0.14–0.33 nmol DW) than in leaves (0.03–0.06 nmol
DW) of control plants (Fig. 5b). Leaf IAA levels were not
significantly affected by exposure to Cu; in roots, the only
significant change was observed in clone B229 with an
almost twofold increase over control levels.
Discussion
In this study, changes in endogenous levels of plant hor-
mones ABA and IAA in response to long-term exposure to
excess soil Cu and its relationship with the accumulation of
Fig. 2 Guaiacol peroxidase
(GPOD) activity (a) and proline
concentration (b) in leaves and
roots of clones M1, B229, and
PE19/66 grown on soil
supplemented (Cu) or not
(Control) with 300 mM CuNO
3
.
Asterisks (*P\0.05,
**P\0.01, ***P\0.001)
indicate significant differences
within each clone between
control and Cu-treated plants
using Tukey’s HSD post hoc
test. Data represent the
mean ±SE (n=3–6)
J Plant Growth Regul
123

protective compounds, PAs and proline, and the activity of
an antioxidant enzyme are addressed for the first time in a
woody species. Results show that there are three distinct
genotype-dependent physiological and biochemical
responses to Cu with potential involvement in the tolerance
strategy of each clone.
Cu Uptake and Organ Allocation are Genotype
Dependent
The amount of Cu that accumulates in roots or that is translo-
cated to leaves and the intensity of its effects on plants generally
depend on plant species and genotype sensitivity, specific
organ, and concentration/duration of metal exposure (Ali and
others 2006;Yruela2009; Cuypers and others 2011). Normal
Cu concentrations in plants are between 1 and 5 lgg
-1
DW
(Marschner 1995), and slightly higher in leaves (5–20 lgg
-1
DW; Baker and Senef 1995). In our study, therefore, leaf Cu
content in Cu-exposed plants was relatively low and cannot be
considered toxic. Conversely, the high metal accumulation in
roots is in accordance with previous studies in poplar, willow,
and birch (Borghi and others 2007; Castiglione and others 2009;
Cuypers and others 2011 Kopponen and others 2001¸Vande-
casteele and others 2005) and reached potentially toxic levels
according to Alloway (1995). The three poplar clones analyzed
exhibited large differences in Cu-uptake capacity, with M1
accumulating the most and B229 the least. Previous studies in
willow and poplar also revealed strong clonal variability in
HM-uptake capacity (e.g., Dos Santos Utmazian and others
2007). Metal allocation also differed between poplar clones,
with M1 showing not only the highest Cu accumulation in roots,
but also greater Cu translocation to leaves, a pattern that could
account for its enhanced antioxidant response (GPOD and PAs;
see below).
Fig. 3 Logarithm of guaiacol
peroxidase (GPOD) activity
plotted against the reciprocal of
endogenous Cu concentration in
leaves (a) and roots (b)of
clones M1, B229, and PE19/66
grown on soil supplemented
(Cu) or not (Control) with
300 mM CuNO
3
J Plant Growth Regul
123

Shoot Growth and Photosynthetic Functionality
as Indicators of Cu Tolerance
Even though application of high Cu concentrations usually
reduces plant growth also in poplar (Borghi and others
2007), the response is genotype dependent (Dos Santos
Utmazian and others 2007; Borghi and others 2008; Gaudet
and others 2011). In the three poplar clones examined here,
exposure for 4 months to Cu treatment did not significantly
affect shoot growth (shoot length and diameter, leaf bio-
mass) nor did it cause visible toxicity symptoms (data not
shown). As previously reported for Arabidopsis and lupin
Fig. 4 Free (a,b,c) and conjugated (d,e,f) polyamine concentra-
tions of clones M1, B229, and PE19/66 grown on soil supplemented
(Cu) or not (Control) with 300 mM CuNO
3
: putrescine (a,d),
spermidine (b,e) and spermine (c,f). Asterisks (*P\0.05,
**P\0.01, ***P\0.001) indicate significant differences within
each clone between control and Cu-treated plants in leaves and roots
using Tukey’s HSD post hoc test. Data represent the mean ±SE
(n=3–5)
J Plant Growth Regul
123

plants exposed to excess Cu, mineral nutrition was likewise
not significantly affected (Mourato and others 2009; Cuy-
pers and others 2011).
Leaf photosynthetic functionality was reduced, albeit to
different degrees, in the three poplar clones grown on Cu-
supplemented soil, and was probably due to diminished
stomatal conductance and photosynthetic pigments,
although mesophyll-related factors, metabolic limitation,
and secondary effects, namely oxidative stress, cannot be
excluded. Indeed, degradation of photosynthetic pigments
in plants exposed to elevated concentrations of HMs has
been widely reported (Kuc
ˇera and others 2008), and Cu can
interfere with the biosynthesis of the photosynthetic
machinery (Lidon and Henriques 1991). A reduction of
photosynthetic gas-exchanges is generally considered a
protective mechanism to safely regulate the activity of the
photosynthetic machinery during abiotic, including HM,
stress (Chaves and others 2009; Maksymiec 2007).
GPOD Activity Correlated with High Cu
Accumulation in Organs
The examined poplar clones responded to potentially toxic
levels of Cu (at least at the root level) by differential
activation of protective mechanisms (antioxidant enzyme
and nonenzymatic compounds). Peroxidases represent one
of the enzymatic detoxification systems that help plants to
avoid the accumulation of ROS upon exposure to HMs
(Van Assche and Clijsters 1990). Indeed, enhancement of
the peroxide scavenging system has been frequently
Fig. 5 Free ABA (a) and IAA
(b) content in leaves and roots
of clones M1, B229, and PE19/
66 grown on soil supplemented
(Cu) or not (Control) with
300 mM CuNO3. Asterisks
(*P\0.05, **P\0.01)
indicate significant differences
within each clone between
control and Cu-treated plants
using Tukey’s HSD post hoc
test. Data represent the
mean ±SE (n=3–5)
J Plant Growth Regul
123

observed in herbaceous species in response to excess Cu
and taken as an indication of its role against oxidative
stress (Mocquot and others 1996; Cuypers and others 2002;
Ali and others 2006; Kim and others 2010). In the three
poplar clones analyzed here, Cu-induced GPOD activity in
roots or leaves (or both, depending on the clone) confirmed
this notion. The dose–response relationship between
enzyme activity and endogenous Cu concentration in both
leaves and roots is consistent with findings in Zea mays
(Lagriffoul and others 1998) and suggests that GPOD
activity could be a reliable indirect biomarker of Cu impact
when morphological parameters are not affected despite
high tissue metal content (Mocquot and others 1996). The
observed increment in GPOD activity in relation to tissue
Cu content was genotype-dependent. Thus, in clone M1,
GPOD activity increased both in leaves and roots, coupled
with higher, as compared with the other clones, Cu accu-
mulation in both organs; in PE19/66, this relationship was
evident only for roots. The induction of GPOD in leaves
and/or roots of the two clones that accumulated high
amounts of Cu at the root level can be explained in terms of
roots being the primary sensing organs and the existence of
long-distance root-to-shoot signaling the importance of
which has been recently outlined (Shabala and others
2016). On the other hand, in B229 the lack of a GPOD
response at either the leaf or root level may derive from the
fact that, in this clone, root Cu accumulation did not reach
the critical threshold value for toxicity estimated for these
poplar plants.
Enhanced PA and Proline Levels in Roots are
a Common Response to Cu Accumulation
Several strategies can be adopted by plants to alleviate HM
toxicity, among which is the modulation in endogenous
levels of PAs (Bhardwaj and others 2014). In the examined
poplar clones, the overall Cu-induced accumulation of total
free and conjugated PAs points to a common response to
excess Cu, as supported also by the strong linear relation-
ship of aboveground changes in PA levels with below-
ground changes (data not shown). PAs seem to play a
protective role in plants exposed to stress by regulating
redox homeostasis (Saha and others 2015), by acting as
metal chelators (Shevyakova and others 2011) or as com-
ponents of the ROS signaling pathway (Pottosin and others
2014), and by activating the plant antioxidant defense
system (Velikova and others 2000). Not surprisingly,
therefore, upregulation of PA metabolism has been previ-
ously reported for poplars exposed to high Cu concentra-
tions under laboratory conditions and in field trials on
contaminated soil (Franchin and others 2007; Lingua and
others 2008; Castiglione and others 2009; Cicatelli and
others 2010). As with other parameters, a differential and
organ-specific accumulation of PAs in response to Cu
occurred among the poplar clones. Interestingly, the
genotype- and organ-specific changes in PA levels in
response to Cu followed a similar trend as those observed
for GPOD activity. Thus, in clone M1, enhanced total free
and conjugated leaf and root PA concentrations were
associated with a strong induction of GPOD in both organs,
whereas in B229 or PE19/66, a similar relationship was
observed only at the leaf or root level, respectively. This
overall relationship between PAs and GPOD activity, and
the fact that both showed a strong dose-dependent rela-
tionship with organ Cu content supports the contention that
PAs might contribute to Cu tolerance by enhancing
antioxidant protection (Velikova and others 2000).
Differences among clones were evident also in the
organ-specific distribution of individual PAs. Free and
conjugated Put responded mainly in leaves of clones M1
and B229, whereas in PE19/66 this diamine accumulated
only at the root level. Positive effects of increased
endogenous contents of Put (Pal and others 2015) and of its
exogenous application on the biochemical responses to Cu
stress (Chen and others 2013) have been reported in vari-
ous plant species. Clone M1, which accumulated the
highest root amount of Cu relative to the other clones,
exhibited the strongest increase in total free PAs in roots,
caused mainly by higher contents of Spd and Spm. The
protective effect of these PAs in mitigating abiotic stress
has been reported. Transgenic pear lines overexpressing
spermidine synthase (SPDS) performed better in the pres-
ence of HMs (Wen and others 2010), whereas antisense
lines with reduced SPDS expression had reduced Spd titers
and exhibited increased growth inhibition under salinity
and Cd stress (Wen and others 2011). Reduced endogenous
Spm in an Arabidopsis mutant was associated with
increased sensitivity to aluminum (Nezames and others
2013). The prevalence of soluble conjugated PAs (cova-
lently linked to phenolics) is consistent with the abundance
of phenylpropanoids in poplar tissues (Tsai and others
2006). These phenylamides are particularly active as free
radical scavengers (Velikova and others 2007). Again,
in clone M1 conjugated Put and, especially, Spd and Spm
root contents increased most dramatically under Cu treat-
ment. Thus, clone M1 may be regarded as the one in which,
despite accumulating the highest amount of Cu, stress was
mitigated based on the protective effect of PAs.
Proline is among the protective compounds that can
quench singlet oxygen as well as hydroxyl radicals that
might result from Cu-catalyzed Fenton chemistry under
excess Cu (Siripornadulsil and others 2002). Similar to
PAs, root proline levels also increased as a function of Cu
concentration in the three poplar clones. In M1 and PE19/
66, the Cu induction of root proline levels was concomitant
with the highest Cu concentrations in this organ. Although
J Plant Growth Regul
123

the precise relationship between the PA and proline sig-
naling pathways remains unclear (Pal and others 2015),
both exert several protective functions (Szabados and
Savoure
´2010) and could account for Cu tolerance in
poplar, especially in these two clones.
IAA and ABA Display Genotype- and Organ-
Dependent Responses to Cu
Exposure of poplar plants to a high dose of Cu in the soil
and tissue accumulation of this metal induced changes in
the endogenous status of plant hormones. These findings
confirm previous studies carried out on herbaceous species
some of which also reported Cu effects on the expression
of genes involved in the biosynthesis, conjugation, and
transport of these hormones (Table 1).
Differential accumulation of ABA in response to Cu
occurred among the poplar clones. In PE19/66 and B229,
enhanced ABA concentrations corroborate previous results
for Cu in herbaceous species (Table 1) and for other metals
(Ashger and others 2015). Accumulation of ABA and
regulation of ABA metabolic gene expression have been
suggested to contribute to Cu tolerance without impacting
growth (Choudhary and others 2012). Exogenous applica-
tion of ABA has also been associated with improved tol-
erance in several plant species exposed to HM stress
(Ashger and others 2015). The increased ABA contents in
PE19/66 and B229 were likely responsible for stomatal
limitation on photosynthesis, whereas a different mecha-
nism seemed to modulate this physiological process in M1
under Cu treatment. In this clone, ABA did not increase,
but leaf Put levels did; in this case, downregulation of
stomatal conductance might be explained by a PA-induc-
tion of other signaling molecules like nitric oxide, a well-
recognized component of drought- and ABA-mediated
stomatal closure (Yamasaki and Cohen 2006).
Interestingly, in roots of Cu-treated poplar plants, an
overall good correlation between proline and ABA
(r
2
=0.65; P\0.05) was observed. In leaves of clone
PE19/66, the concomitant strong increases in ABA con-
centration and proline content upon Cu treatment also
appears to support the regulation of proline biosynthesis
by ABA in response to abiotic stress (Xiong and others
2001; Shevyakova and others 2013). Indeed, ABA-in-
duced signaling may trigger expression of stress-related
genes followed by synthesis of protective compounds
such as proline (Kavi Kishore and others 2005). ABA
accumulation in response to osmotic stress has been
shown to regulate expression of P5CS, the gene encoding
for an enzyme involved in proline biosynthesis (Xiong
and others 2001). Moreover, a role of ABA in HM tol-
erance via regulation of the antioxidant machinery, as
suggested by recent findings in Vigna radiata after
exogenous application of ABA under Cd stress (Li and
others 2014), cannot be excluded. Finally, Shevyakova
and others (2013) suggested that ABA coordinates the
intracellular levels of both PAs and proline.
Differences in sensitivity of poplar genotypes to abiotic
stress often involve changes in auxin physiology (Tognetti
and others 2012, and references therein). Although
decreased auxin content has been observed and considered
an adaptive mechanism under adverse environmental con-
ditions in poplar (Popko and others 2010), IAA home-
ostasis has been suggested to mediate plant tolerance to
metal stress (Pasternak and others 2005; Elobeid and Polle
2012). Thus, it is plausible that in the examined poplar
clones, maintaining the aboveground auxin pool at physi-
ological levels optimal for plant performance may account
for the lack of Cu-induced reduction in shoot growth after
long-term exposure to Cu. This observation is consistent
with recent findings in Arabidopsis suggesting that Cu-
induced expression of auxin homeostasis-related genes and
auxin accumulation might be responsible for ensuring
normal plant growth and development under this stress
condition (Wang and others 2015). Cu-induced growth
reduction, on the other hand, has been associated with an
increase in the activity of enzymes implicated in auxin
degradation (Table 1). In poplar, downregulation of both
growth and photosynthetic functionality in response to Cd
stress were associated with the increased activity of an
enzyme involved in IAA conjugation with the probable
consequent depletion of active auxins (Elobeid and others
2012).
Present results indicate that the only substantial change
in IAA was an increase in roots of clone B229, confirming
previous findings in Arabidopsis and other herbaceous
species (Table 1; Bankaji and others 2014) and pointing to
an organ- and genotype-dependent response to Cu. Chan-
ges in IAA levels and auxin-related enzymes involved in
the response of poplar to Cd were also shown to be organ-
specific (Elobeid and others 2012). Increased IAA in B229
may account for the less dramatic reduction of gas
exchanges observed in this clone in spite of higher leaf
ABA levels. In fact, auxin promotes stomatal opening
(Lohse and Hedrich 1992), thus counteracting ABA-in-
duced closure. Exogenous application of IAA improved
Cu-induced downregulation of photosynthetic functionality
in Helianthus annuus (Ouzounidou and Ilias 2005).
One may speculate on the possibility that in clone B229
this enhanced root IAA pool may promote the induction
and growth of lateral roots as a contributing factor in stress
tolerance (Lequeux and others 2010; Pet}
o and others 2011;
Vitti and others 2014). In fact, increased root branching
could be considered as a tolerance strategy aimed at redi-
recting root growth toward soil zones containing less
metals (Vitti and others 2014).
J Plant Growth Regul
123

Conclusions
Taken together, the present results show that under long-
term exposure to excess Cu in the soil and potentially toxic
Cu levels in the roots, poplar plants activated several
mechanisms depending on the clone: (1) enhanced
antioxidant enzyme (GPOD) activity, and accumulation of
PAs, possibly responsible for reducing the impact of Cu-
induced oxidative damage to the photosynthetic apparatus,
thereby maintaining photosynthetic functionality at levels
compatible with growth; (2) modulation of the plant hor-
mone pools, which could be related to other processes,
such as proline production, regulation of stomatal control
on photosynthesis, and growth. Results suggest that GPOD,
proline, PAs, IAA, and ABA are integrated in the response
and tolerance of poplar genotypes to grow and develop
under Cu stress in a clone-specific manner and in relation
to Cu-uptake capacity (Fig. 6). The ability of clone M1 to
cope with high amounts of HMs in plant organs (true tol-
erance according to Baker 1981; Maestri and others 2010)
seems to rely on the activation of the enzymatic and
nonenzymatic antioxidant systems (GPOD and PAs) that
could contribute to reduce the impact of Cu-induced
oxidative damage. Conversely, the tolerance strategy of
clone PE19/66, a strong Cu accumulator at the root level
but lacking an increased antioxidant protection by PAs and
GPOD, seems to rely on the protective capacity of
enhanced proline and ABA levels in both organs. The
overall response of clone B229 may reveal an avoidance
strategy (Maestri and others 2010) requiring attenuated
antioxidant defenses, namely low Cu accumulation,
accompanied by hormonal (IAA) changes that potentially
lead to root growth modifications suitable for avoiding
excess soil metal.
Our findings warrant further investigation on the inter-
actions between ROS signaling and antioxidant defense
and plant hormones as part of a large and complex network
that modulates short- and long-term responses of poplar to
excess HMs. Further understanding of the specific roles
that hormones play in environmental adaptation to HMs
could lead to the development or selection of genotypes
better adapted to stressful environments.
Acknowledgments This study was supported by STSM Grants from
COST Action FP0903 ‘‘Climate Change and Forest Mitigation and
Adaptation in a Polluted environment (MAFor)’’ and by COST
Action FP1106 ‘Studying Tree Responses to extreme Events: a
SynthesiS (STReESS)’ to MK. Additional support was provided by
funds (Ricerca Fondamentale Orientata, RFO 2012) from the
University of Bologna to SB and by the Ministry of Education, Sci-
ence and Technological Development of the Republic of Serbia,
Project No. III 43002—Biosensing technology and the global systems
for continuous research and integrated management of ecosystems to
SO and MK.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
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