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Mixed heavy metal stress on photosynthesis,

transpiration rate, and chlorophyll content in poplar
hybrids
a a
Romika Chandra & Hoduck Kang
a
Department of Biological and Environmental Science, Dongguk University, Biomedi
Campus, 32 Dongguk-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do 410-820, Republic of
Korea
Published online: 19 Jun 2015.

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rate, and chlorophyll content in poplar hybrids, Forest Science and Technology, DOI: 10.1080/21580103.2015.1044024

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 Forest Science and Technology
2015, 1 7

Mixed heavy metal stress on photosynthesis, transpiration rate, and chlorophyll content in
poplar hybrids
Romika Chandra and Hoduck Kang*
Department of Biological and Environmental Science, Dongguk University, Biomedi Campus, 32 Dongguk-ro, Ilsandong-gu, Goyang-si,
Gyeonggi-do 410-820, Republic of Korea

(Received 14 April 2015; Accepted 20 April 2015)

The present work aimed to study the physiological effects of mixed heavy metals, cadmium (Cd), copper (Cu), chromium
(Cr), and zinc (Zn) in four poplar hybrids. Poplar hybrids were treated once with six different concentrations (0, 5, 50, 100,
200, and 500 ppm) of mixed heavy metals for a period of 3 months. The photosynthetic and transpiration rates were
measured before and during treatment using an LCi SD portable photosynthesis system whereas the photosynthetic
pigments were analytically determined. According to the results obtained, poplar hybrids exhibited a decrease in
chlorophyll content and carotenoids except for hybrid 1 (Eco 28) which showed an increase at 200 and 500 ppm heavy
metal concentrations. The photosynthetic rate decreased with increasing heavy metal concentrations; however, an increase
was observed at the highest concentration of 500 ppm at 14.54 mmol m¡2 s¡1. Transpiration rates were significantly
affected by increased heavy metals. Hybrid 1 (Eco 28) was deduced as the hybrid that has the ability to tolerate high levels
of mixed heavy metals. Among the four poplar hybrids tested, hybrid 1 (Eco 28) was the best candidate for
phytoremediative experiments under greenhouse conditions. These results are valuable in understanding how various tree
hybrids respond to mixed heavy metal stress, especially when using them as bio indicators for phytoremediation
Forest Science and Technology

experiments.
Keywords: poplar hybrids; mixed heavy metals; photosynthetic rate; transpiration rate; photosynthetic pigments

Introduction and disturbing photosynthesis. Inhibition of photosynthesis

Heavy metal contamination of the environment from vari-
ous sources, including industry and agriculture, is an area of
concern around the world. Although heavy metals are natu-
ral components in soils at trace levels, various anthropo-
genic activities such as increased environmental pollution
from industry, agriculture, and mining have contributed to
undesirable toxic accumulations of heavy metals.
Phytoremediation is a cleanup technology for metal
contaminated soils. Some model phytoremediators
include various varieties of trees; examples include cot-
tonwood, poplar, and willows. Trees are ideal in the reme-
diation of heavy metals because they can withstand higher
concentrations of pollutants due to their large biomass
(Paz-Alberto and Sigua 2013). They have the ability to
accumulate large amounts of the contaminants in their
systems because of their size and extensive roots systems.
Furthermore, trees have the ability to stabilize an area,
prevent erosion, and minimize the spread of the contami-
nants. They can be easily harvested and removed from the
area with minimal risk, allowing the effective removal of
contaminants (Paz-Alberto and Sigua 2013).
Some heavy metals such as copper (Cu), zinc (Zn),
cobalt (Co), and iron (Fe) are essential in trace amounts
for various metabolic activities in plants. However, excess
of any kind of metal (essential or non-essential) adversely
affects plant metabolism (Hall 2002). In plants, metals
exert their toxic action mostly by damaging chloroplasts
is the consequence of interference of metal ions with pho-
tosynthetic enzymes and chloroplast membranes (Aggar-
wal et al. 2012). In higher plants, photosynthesis is
indirectly reduced by heavy metal accumulation in leaves
which influences the functioning of the stomata and hence
affects photosynthesis and transpiration rates overall.
Photosynthetic pigment reduction by heavy metals affects
photosynthesis indirectly, hence the use of non-destructive
methods and the ease of measurement allows photosyn-
thetic pigments to be frequently used to determine stress
for regulatory purposes (Aggarwal et al. 2012).
The sensitivity of plants to heavy metals depends on
an interrelated network of physiological and molecular
mechanisms such as: (i) uptake and accumulation of met-
als through binding of extracellular exudates and cell wall
constituents; (ii) efflux of heavy metals from cytoplasm to
extra nuclear compartments including vacuoles; (iii) com-
plexation of heavy metal ions inside the cell of various
substances, for example, organic acids, amino acids, phy-
tochelatins, and metallothioneins; (iv) accumulation of
osmolytes and osmoproteins and induction of antioxida-
tive enzymes; and (v) activation or modification of plant
metabolism to allow adequate functioning of metabolic
pathways and rapid repair of damaged cell structures
(Cho et al. 2003).
Various poplar genotypes have been used to determine
their responses at physiological and molecular levels in

*Corresponding author. Email: hdk0225@dongguk.edu

ISSN 2158-0103 print/ISSN 2158-0715 online

Ó 2015 Korean Forest Society
http://dx.doi.org/10.1080/21580103.2015.1044024
http://www.tandfonline.com
 2 R. Chandra and H. Kang
different heavy metals. Sebastiani et al. (2004) looked at
the effects of heavy metal (Zn, Cu, chromium [Cr], and
cadmium [Cd]) enriched organic waste on biomass por-
tioning and heavy metal accumulation in plant organs in
two poplar clones (Populus deltoids x maximowiczii
clone Eridano and P. x euramericana clone I-214).
Gaudet et al. (2011) compared the physiological and
molecular responses of Cd stress in two Populus nigra L.
genotypes originating from contrasting environments.
Robinson et al. (2007) investigated the potential of hybrid
poplar for boron (B) phytomanagement using a lysimeter
experiment and a field trial on B contaminated wood
waste. Zacchini et al. (2011) studied the effects of Cd
accumulation and tolerance in P. nigra and Salix alba.
Despite numerous studies, the specific mechanisms
involved in heavy metal accumulation and detoxification
are yet to be well described. Characterization of these
mechanisms could be important in improving the potential
of Salicaceae plants for phytoremediation.
The main aims of this study were to use non-destruc-
tive and easy access parameters to determine the effects
of mixed heavy metals (Cd, Cr, Cu, and Zn) stress, under
greenhouse conditions, on: (i) leaf chlorophyll a (chl a),
chlorophyll b (chl b), total chlorophyll, chlorophyll a/b
ratio, and carotenoids; and (ii) photosynthesis and transpi-
Forest Science and Technology
ration rates.
Materials and methods
Plant materials and experimental conditions
The experiment was conducted under greenhouse condi-
tions in Ilsan, located northwest of Seoul, South Korea
during the months of April September 2014. Four Popu-
lus hybrids were selected based on genetic diversity,
growth, and wellbeing in early spring 2014. The details of
the clones are provided in Table 1. Cuttings of approxi-
mately 15 cm lengths from the four hybrids were planted
in 2 L pots filled with sandy loam soil in which the origi-
nal plants were growing in the field. After 2 months to
allow sprouting and roots to stabilize, the plantlets were
treated with heavy metals of various concentrations rang-
ing from 0 ppm as control followed by 5, 50, 100, 200,
and 500 ppm. Mixed heavy metals utilized were chro-
mium III chloride (CrCl3), copper (II) chloride (CuCl2),
cadmium chloride (CdCl2), and zinc chloride (ZnCl2).
Each heavy metal was prepared separately and equal
amounts of the same concentrations were mixed for treat-
ment. Plantlets were treated with 20 ml of mixed heavy
metal per plantlet once only. Plantlets were regularly
Table 1. Poplar hybrids selected for greenhouse phytoremedia-
tive experiment.
Code Hybrid name
1 Eco 28 (Populus euramericana Guinier)
2 DN 034 (Populus deltoids x P. nigra)
3 TN 074 (Populus tricocarpa x P. nigra)
4 TD 225 (Populus tricocarpa x P. deltoids)
watered over the 3 month treatment period. The treated
experimental pots were set up according to a complete
randomized block design (CRBD). Each treatment was
replicated six times and appeared once in each block.
Photosynthesis and transpiration rates
An LCi SD (ADC Bioscientific Ltd., Hoddesdon, UK)
portable photosynthesis system was used to measure the
photosynthetic and transpiration rates of the plants, which
were recorded before treatment and 2 months after treat-
ment to determine any changes that might occur over time.
Photosynthetic pigments
To estimate photosynthetic pigments chl a, chl b, and car-
otenoids, plant materials (leaves) were harvested from the
greenhouse and 0.1 g was placed in 8 ml of 80% acetone
for 7 days under dark conditions in the refrigerator. After
7 days absorbances were taken with UV/VIS spectropho-
tometer at 663, 645, and 470 nm (Skanlt software 3.2 for
Multiskan Co., Thermo Fisher Scientific, Waltham, MA,
USA). Chlorophyll content was estimated according to
Arnon (1949) and carotenoid content according to Lich-
tenthaler (1987).
Statistical analysis
Collected data are expressed as mean § SEM. All data
were analysed by the Holm-Sidak ANOVA multiple com-
parison statistics using GraphPad Prim 6.0 software.
Statistical significance was accepted at p ˂ 0.05.
Results
Photosynthesis and transpiration rates
The exposure of mixed heavy metals to poplar hybrids
under greenhouse conditions resulted in an increase in
photosynthetic rate with increase in heavy metal concen-
trations (Table 2). Hybrid 1 had the highest photosynthetic
rate of 14.54 mmol m¡2 s¡1 at 500 ppm of mixed heavy
metal concentration, being the highest photosynthetic rate
among all four hybrids. No other significant differences
were observed among other heavy metal concentrations
for hybrid 1. For hybrid 2, fluctuations in photosynthetic
rate were observed across the concentrations; however,
the lowest photosynthetic rate of 7.75 mmol m¡2 s¡1 was
observed at 5 ppm mixed heavy metal concentration and
the highest of 12.60 mmol m¡2 s¡1 at 200 ppm. Hybrid 3
had the lowest photosynthetic rate of 2.61 mmol m¡2 s¡1
at 200 ppm which increased 5 times to 10.08 at 500 ppm.
However, hybrid 4 had a significantly higher photosyn-
thetic rate at 5 ppm of 8.20 mmol m¡2 s¡1 which
decreased to 6.84 and 7.23 for 50 and 100 ppm, respec-
tively. A clear pattern can be observed for all four poplar
hybrids with the photosynthetic rate increasing with
increased mixed heavy metal concentrations, the majority
being significant around 200 500 ppm mixed heavy
metal concentrations.
 Forest Science and Technology 3

Table 2. Photosynthetic rates of four poplar hybrids under mixed heavy metal (Cd, Cr, Cu, and Zn) treatment in greenhouse conditions.

Photosynthesis rate A (mmol m¡2 s¡1)

Mixed heavy metal (ppm) Hybrid 1 (Eco 28) Hybrid 2 (DN 034) Hybrid 3 (TN 074) Hybrid 4 (TD 225)

0 10.21 § 0.20 10.12 § 0.26 9.05 § 0.34 9.05 § 0.17

5 8.82 § 1.24 7.75 § 0.26 9.72 § 0.27 8.20 § 0.54
50 11.32 § 0.18 11.43 § 0.52 7.11 § 0.42 6.84 § 0.63
100 8.56 § 0.23 10.21 § 0.93 8.12 § 0.46 7.23 § 0.50
200 8.71 § 0.21 12.60 § 0.52 2.61 § 0.50 8.88 § 0.53
500 14.54 § 0.70 11.73 § 0.11 10.08 § 0.20 7.70 § 0.56

Note: Mean values § SEM.


p < 0.0001;  p < 0.001;  p < 0.01;  p < 0.05. Values significantly different from control (0) (n D 10).

Transpiration rates varied across the hybrids, the high-
est value coinciding with 50 ppm for hybrid 1 at
11.23 mmol m¡2 s¡1 followed by 500 ppm at 8.89 mmol
m¡2 s¡1 (Table 3). Heavy metal concentrations of 5 ppm
and 200 ppm had significant transpiration rates when com-
pared to control (0 ppm). Hybrid 2 had the highest transpi-
ration rate at 9.99 mmol m¡2 s¡1 at 200 ppm followed by
9.61 at 50 ppm. Transpiration rates significantly different
from control were at the low mixed heavy metal concen-
trations of 5 ppm and 100 ppm. The transpiration rates of
Forest Science and Technology
hybrid 1 showed a general decreasing trend for chl a, chl
b, and total chlorophyll, with increasing concentrations of
heavy metals up to 200 ppm after which the chlorophyll
contents increased. The initial decrease in pigment con-
tents chl a and total chlorophyll were highly significant.
However, the carotenoid contents decreased with increas-
ing heavy metal concentrations and at 500 ppm increased
dramatically to 3.17 mg/g, although the increase was not
significant. In hybrid 2 and hybrid 3 the chl a, chl b, and
total chlorophyll contents, as well as carotenoid contents,

hybrid 3 were most significant at all concentrations. The
highest of 10.87 mmol m¡2 s¡1 at 50 ppm followed by
10.14 mmol m¡2 s¡1 at 100 ppm and the lowest of
3.42 mmol m¡2 s¡1 at 200 ppm. The lowest transpiration
rates were observed in hybrid 4 where the apparent lowest
transpiration rate was at 50 ppm (3.55 mmol m¡2 s¡1) and
the highest was at 200 ppm (8.14 mmol m¡2 s¡1). No spe-
cific relationship was observed for transpiration rates
across the different mixed heavy metal concentrations in
hybrid 4. Significant transpiration rates were observed
across all four poplar hybrids. However, the differences
varied for each hybrid; in general, a decrease in transpira-
tion rate was observed with an increase in mixed heavy
metal concentrations.
Photosynthetic pigments
The exposure of poplar hybrids to heavy metals for phy-
toremediation greenhouse experiments overall resulted in
significant reductions in chlorophyll and carotenoid con-
tents in leaves (Table 4). The chlorophyll content in
decreased as compared to control. This suggests that the
various concentrations of heavy metals affect the pig-
ments contents. The most significant change in leaf chl a,
chl b, and carotenoid contents was observed in hybrid 4 at
500 ppm of 9.00 mg/g, 3.71 mg/g and 1.45 mg/g respec-
tively, being the lowest significant values across all
hybrids. Overall, slight decreases in chl a and b were
observed; however, 500 ppm heavy metal concentration
had the lowest photosynthetic pigments in hybrid 4.
There were no significant changes in chlorophyll a/b
ratio across all concentrations in all four hybrids (results
not shown). An overall decrease in carotenoid content
was observed for all poplar hybrids; however, no signifi-
cant changes were observed in leaf carotenoid contents in
all hybrids across all heavy metal concentrations.
Discussion
Heavy metal stress alters many physiological and meta-
bolic processes in plants. The data presented demonstrate

Table 3. Transpiration rates of four poplar hybrids under mixed heavy metal (Cd, Cr, Cu, and Zn) treatment in greenhouse conditions.

Transpiration rate E (mmol m¡2 s¡1)

Mixed heavy metal (ppm) Hybrid 1 (Eco 28) Hybrid 2 (DN 034) Hybrid 3 (TN 074) Hybrid 4 (TD 225)

0 7.45 § 0.24 10.17 § 0.41 8.29 § 0.39 5.99 § 0.16

5 5.96 § 0.70 8.64 § 0.52 9.96 § 0.18 6.38 § 0.15
50 11.23 § 0.05 9.61 § 0.26 10.87 § 0.44 3.55 § 0.53
100 7.84 § 0.28 8.77 § 0.78 10.14 § 0.72 6.24 § 0.61
200 5.30 § 0.11 9.99 § 0.38 3.42 § 0.53 8.14 § 0.34
500 8.89 § 0.13 7.64 § 0.14 9.53 § 0.28 7.52 § 0.47

Note: Mean values § SEM.


p < 0.0001;  p < 0.001;  p < 0.01;  p < 0.05. Values significantly different from control (0) (n D 10).
 4 R. Chandra and H. Kang

Table 4. Mixed heavy metal (Cd, Cr, Cu, and Zn) treatment effects on photosynthetic pigment concentrations in four poplar hybrids.

Photosynthetic pigments (mg/g)

Hybrid Mixed heavy metal (ppm) chl a chl b chl (aCb) Carotenoid

Eco 28 0 16.08 § 0.98 6.65 § 0.43 22.61 § 1.31 2.77 § 0.12

5 13.21 § 1.48 5.50 § 0.67 18.71 § 2.14 2.52 § 0.26
50 13.62 § 1.50 5.48 § 0.78 19.10 § 2.27 2.60 § 1.89
100 13.44 § 1.21 5.52 § 0.53 18.96 § 1.74 2.46 § 0.12
200 14.39 § 2.60 5.94 § 1.11 20.33 § 3.71 2.66 § 0.42
500 17.84 § 1.89 5.54 § 1.06 23.38 § 1.40 3.17 § 0.32
DN 034 0 17.34 § 1.07 7.03 § 0.70 24.48 § 1.69 3.34 § 0.18
5 17.50 § 0.97 7.55 § 0.54 25.05 § 1.51 3.18 § 0.20
50 14.20 § 1.22 6.25 § 0.72 20.46 § 1.61 2.87 § 0.23
100 16.67 § 0.91 6.85 § 0.41 23.51 § 1.31 3.12 § 0.20
200 15.69 § 0.60 6.41 § 0.36 22.11 § 0.95 2.91 § 0.08
500 14.08 § 0.80 5.56 § 0.34 19.63 § 1.14 2.77 § 0.13
TN 074 0 18.07 § 1.03 7.80 § 0.51 25.87 § 1.53 3.73 § 0.39
5 15.81 § 1.08 6.76 § 0.51 22.56 § 1.58 2.89 § 0.27
50 15.26 § 0.80 6.41 § 0.35 21.68 § 1.15 2.91 § 0.17
100 15.71 § 1.11 6.64 § 0.55 22.34 § 1.66 3.06 § 0.60
200 15.70 § 0.76 6.64 § 0.28 22.44 § 0.98 2.83 § 0.12
500 15.90 § 0.73 6.86 § 0.37 22.94 § 1.14 2.81 § 0.16
TD 225 0 15.79 § 1.17 6.76 § 0.58 22.55 § 1.75 2.75 § 0.20
5 14.98 § 1.30 6.35 § 0.64 21.29 § 1.92 2.46 § 0.16
Forest Science and Technology

50 14.24 § 1.32 6.12 § 0.75 19.80 § 1.76 2.23 § 0.19

100 13.43 § 0.91 5.63 § 0.42 19.06 § 1.33 2.20 § 0.15
200 14.72 § 0.37 6.20 § 0.17 20.92 § 0.54 2.42 § 0.06
500 9.00 § 0.74 3.71 § 0.33 12.46 § 1.14  1.45 § 0.13

Note: Mean values § SEM.


p < 0.0001; p < 0.001; p < 0.01; p < 0.05. Values significantly different from control (0) (n D 4).

that mixed heavy metal exposure leads to significant
decreases in photosynthetic pigments in poplar hybrids.
Chlorophyll content is often measured in plants to assess
the impact of environmental stress, as changes in pigment
content are linked to visual symptoms of plant illness and
photosynthetic productivity (Parekh et al. 1990).
In the present investigation, the photosynthetic rates
decreased for all poplar hybrids across all heavy metal
concentrations; however, only hybrid 1 (Eco 28) had a
high photosynthetic rate at 500 ppm (Figure 1). Decline in
photosynthetic rate has been exhibited in other plants, due
to the reduction in photosynthetic pigments by the heavy
metals. Heavy metals such as mercury (Hg), Cu, Cr, Cd,
and Zn have been found to decrease the chlorophyll con-
tent in various plants in most cases (Aggarwal et al. 2012).
This decline in photosynthetic pigments is most probably

Figure 1. Effects of mixed heavy metal treatment on photosynthetic rate of four poplar hybrids under greenhouse conditions.
 Forest Science and Technology 5

Figure 2. Effects of mixed heavy metal treatment on transpiration rate of four poplar hybrids under greenhouse conditions.
Forest Science and Technology

Figure 3. Effects of mixed heavy metals on chl a, chl b, total chlorophyll (aCb), and carotenoid contents in leaves of the four poplar
hybrids at different heavy metal concentrations. Values are mean § SEM, significance at p ˂ 0.05 (n D 4).
 6 R. Chandra and H. Kang
due to the inhibition of the reductive steps in the biosyn-
thetic pathways of photosynthetic pigments due to the high
redox potential of many heavy metals. In addition, the key
enzyme, protochlorophyllide reductase, which is involved
in the reduction of protochlorophyll to chlorophyll is well
known to be inhibited by heavy metals (De Filippis and
Pallaghy 1994). Various authors have reported similar
decreases in chlorophyll content under heavy metal stress
in cyanobacteria, unicellular chlorophytes (Chlorella),
gymnosperms such as Picea abies and angiosperms, such
as Zea mays, Quercus palustrus and Acer rubrum, sun-
flower as well as almond (Siedlecks and Krupa 1996; Zen-
gin and Munzuroglu 2006; Elloumi et al. 2007). A few
reports show an enhancement of pigments after exposure
to heavy metals (Devi Prasad and Devi Prasad 1982).
The contradictory segments of the data are probably due
to the effects of mixed heavy metals, which compete with
each other in the soil water medium. Uptake by the roots
and their levels of accumulation in the leaves vary depend-
ing on the hybrid, hence showing an overall expected
decline in hybrids 2, 3, and 4. Whereas hybrid 1 had the
highest photosynthetic rate, decreases for 5, 50, and
100 ppm were observed when compared to control; how-
ever, an increase higher than control is observed at 500 ppm
heavy metal concentration. The significant increase in photo-
Forest Science and Technology
synthetic and transpiration rates (Figure 2) for hybrid 1 are
also supported by the increase in photosynthetic pigments
(Figure 3). The overall biological accumulation coefficient
of heavy metals in the plant would be a contributing factor
in terms of explaining how hybrid 1 overall responds to
high concentrations of mixed heavy metals. This points to
the need for a better understanding of the form of heavy
metal ions in the soil solution, their interaction with the plant
roots, their eventual absorption into the system, and, finally,
their translocation to aboveground parts.
Various authors have studied the effects of single heavy
metals on different plant species. The effects of Cd and lead
(Pb) on Brassica juncea L. exhibited a decline in growth,
chlorophyll content, and carotenoids; however Cd was
found to be more detrimental than Pb (John et al. 2009).
According to the study of physiological effects of Cd and
Cu on pea (Pisum sativum), photosynthetic pigments and
photosynthetic rates declined at all concentrations of Cd
and at high Cu concentrations (Hattab et al. 2009).
Heavy metals such as Cu and Zn are essential for nor-
mal plant growth and development since they are constitu-
ents of many enzymes and other proteins. However,
elevated concentrations of any heavy metal either essential
or non-essential can lead to toxic symptoms and growth
inhibition in most plants. This decrease in plant photosyn-
thetic efficiency could be partly responsible for the overall
decrease in plant growth and biomass production.
Conclusion
In conclusion, it can be deduced that the short term expo-
sure of poplar hybrids to mixed heavy metals does seri-
ously affect the overall photosynthetic pigments and
photosynthesis, as investigated by the studied parameters.
This confirms that the use of photosynthetic parameters
provide helpful information on plant response to mixed
heavy metal exposure. Hybrid 1 (Eco 28), which is a local
hybrid, had higher chl a, carotenoid content, and photo-
synthetic rate at the highest mixed heavy metal concentra-
tion of 500 ppm, which probably suggests that further
investigation needs to be conducted with this hybrid in
order to understand the mechanisms involved that could
make it a suitable candidate for phytoremediative field
work. These results, together with the fact that Populus
euramericana hybrids have been found to be suitable can-
didates for earlier phytoremediative work suggests that
there are probably certain genetic factors that contribute
to higher tolerance of P. euramericana hybrids when
compared to other poplar hybrids.
Disclosure statement
No potential conflict of interest was reported by the authors.
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