INT. J. BIL. BIOTECH., 17 (2): 251-263, 2020.

AN EVALUATION PHYTOREMEDIATION EFFICIENCY OF CARROT PLANTS FOR

HEAVY METAL CONTAMINATED SOILS

Aisha Tabassum and Zamin Shaheed Siddiqui*

Stress Physiology and Phenomic Center, Department of Botany, University of Karachi, Karachi-75270, Pakistan
*Corresponding Author: [email protected]

ABSTRACT

The present study intended to evaluate the remediation ability of carrot plants to remove Cu and Zn from the
contaminated soil using growth and biochemical attributes. Furthermore, the possible mechanisms such as
accumulation or exclusion regarding metal tolerance in carrot plants were also assessed. Our data showed that metal
toxicity affected the plants. However, the growth of root was decreased substantially as compared to shoot under heavy
metal stress. The oxidative stress indicators such as H2O2 and Malondialdehyde (MDA) were increased as the metal
concentration increased. Physiological mechanism enlightened that the level of soluble sugars and chlorophyll content
index was reduced as the metal concentration increased in the soil due to damages in the reactive centers of PSII at
higher metal concentrations, Cu in particular. Furthermore, higher metal content was observed in roots as compared to
shoots. Greater bioaccumulation factor and lower translocation factors were recorded for the carrot plants. The zinc
metal showed relatively better physiological activities due to less metal accumulation in shoot. It is concluded that
carrot plants tolerate Cu and Zn by accumulation of these ions in their roots so that this plant can be better choice for
metal contaminated soil.

Key words: Phyto-remediation, carrot, heavy metal, oxidative stress, copper and zinc.

INTRODUCTION

The term heavy metal is applied to the trace metals up to 1000 ppm or less in earth’s crust having a density
greater than 5000 mg cm-3 (Wijayawardena et al., 2016). Among these metals copper (Cu), zinc (Zn), cadmium
(Cd), nickel (Ni), chromium (Cr), cobalt (Co), mercury (Hg) and lead (Pb), are well reported (Zacarias et al., 2012).
It was suggested that heavy metals are biologically essential at low concentrations for normal metabolic processes of
plants (Diacono and Montemurro, 2010). However, non-essential elements like Pb, Hg, Cd and Sn are not required
for normal metabolism. In nature, both essential and non-essential elements enter the plants but the tolerance
capacities of the plants vary regarding concentration of metal and plant cultivars (Gupta et al., 2010).
Contamination of soil air and water by heavy metals is a serious threat to the environment. Metal toxicity is of
great concern particularly for developing countries such as Pakistan where pressure on agriculture output and
industrial production causes the heavy metals accumulation in the environment (Savvas et al., 2010; Pehluvan et al.,
2012; Wijayawardena et al., 2016). The sources of heavy metals in the land are inorganic and organic fertilizers,
sewage sludge, effluents from various industries, and pesticides; particularly fungicides (Gupta et al., 2010).
Consequently, the high concentrations of the heavy metals in soil become toxic to natural flora and fauna affecting
physiological and biochemical processes (Kovacik et al., 2009; Hediji et al., 2015). The visible and non-specific
symptoms of metal toxicity include rapid inhibition of seed germination, seedling and root growth stunted plants,
chlorophyll degradation and enzyme inhibition (Clemens, 2006; Ali et al., 2013; Gangwar et al., 2014). In cell, most
of the metals such as Cd, Ni, Pb may participate in most of the biochemical pathways which results in the
formation of reactive oxygen species (ROS), which causes oxidative damage in plants (Sharma and Dietz, 2009;
Gill and Tteja, 2010).
Nevertheless, the heavy metals tolerance in plants is manifested by an interaction of a genotype to its
environment (Gallego et al., 2012). Thus, the visible injury symptoms in the form of morphological disorders
regarding germination and growth can be used as predictors to assess the degree of metal tolerance. Some plant
species have evolved adapting means involved in general homeostasis that may enable them to thrive on
metaliferrous soils (Peralta-Videa et al., 2009). Plants also possess a range of cellular mechanisms starting from
detoxification to tolerance against heavy metal stress. In this regards enzymatic i.e. peroxidases (POD), catalases
(CAT), superoxide dismutases (SOD,) and non-enzymatic (proline, carotenoids and sugar accumulation etc.)
defense systems to cope with oxidative damage are well known (Szabados et al., 2011; Shavalli Khan et al., 2014;
Szollosi, 2014).
252 AISHA TABASSUM ET AL.,

Strategically, plants can be grouped as either accumulators or excluders for their metal tolerance mechanism
(Chung et al., 2011). In this way a technology is evolved in which plants can eliminate, sequester or destroy
contaminants from water, soil and air called phyto-remediation (Nakbanpote et al., 2016). This technique is getting
great attention in recent years because of significant variability in plant species for heavy metal tolerance based on
various tolerance strategies (Sytar et al., 2016; Jan et al., 2016).
Daucus carota L. (carrot) is a root vegetable, usually orange in color and are domesticated form native to
southwestern Asia and Europe. Carrot is grown in vast areas of Pakistan. Though, in modern research phyto-
remediation ability of dissimilar plant species becoming major focus but some of the important crop plants still
need to be closely monitored (Mahmood et al., 2005). Further PSII photosynthetic efficiency in metal tolerance
plants is rather scarce (Dixit et al., 2015). Therefore, essential workability of PS II about crop plant species is
lacking in relation to their growth on metal contaminated soils. Therefore, it was imperative that the impacts of
heavy metal toxicity on the plant growth and physiological responses be studied and localizes the metals within the
plant. While considering the impetus for the identification of metal tolerant crops, the present study was being
carried out to explore the cultivation potential of carrot plants for tolerance to Cu and Zn contamination and
physiological information regarding metal tolerance is also provided.

MATERIALS AND METHODS

Collection of plant materials

The plant material/germplasm was obtained from the “Agricultural department University of Karachi”
Experiment was carried out using soil culture (after texture determination) in plastic pots under natural
environmental conditions. Two metals (Cu and Zn) and their combination (Cu+Zn) were used to treat the plants.
Salts of Zn (ZnSO4) and Cu (CuSO4) were used as a source of these metals. Three levels of heavy metal treatments
i.e. 20, 40, and 60 mg kg-1 of soil were applied apart from control. The experiment was carried out in a completely
randomized block design for eight weeks and replicated three times.

Metal uptake by plant modules

The diverse biochemical attributes included metal uptake by various plant modules were calculated by using
Atomic Absorption Spectrometry. In our study, a simple and quick method of digestion of plant modules was used
(Huang et al., 2004). The samples were taken in 50 mL polypropylene tubes with 3.2 mm ventilation holes in their
caps. A microwave digestion system was used for the purpose of digestion. The process comprised of two phases. In
the first step, digestion of plant material with HNO3 is carried out followed by heating the mixture at 75 ˚C (10
minutes) and 109 °C (15 minutes). The second phase comprised of cooling the mixture for ten minutes and after that
100 μL of H2O2 was added. Later the sample was heated again at 109 ˚C for 15 more minutes.

Metal tolerance index (TI), Bio-concentration factor (BCF) and Translocation factor (TF)
Metal tolerance index (Wang et al., 2014) used to predict the sensitivity of tissues to stress induced by heavy
metals. Calculations were also carried out for bio-concentration factor (BCF) to evaluate the phytoremediation
potential of target plant. BCF is the ratio of soil and plant concentration of Cu and Zn (Wang et al., 2014). To find
out metal concentration in shoots/roots the method of Majid et al. (2012) was followed.

Chlorophyll fluorescence and Chlorophyll contents index

The chlorophyll fluorescence emissions were quantified using 8 weeks old dark adapted (30min) leaves. The
pulse amplitude modulation mode of the fluorescence monitoring system (Handy PEA) was used for this purpose.
Initial measurements were recorded under modulated beam of far-red light (LED typical peak at 735 nm
wavelength). The maximum (Fm), original (F0) and fluorescence yields were observed at weak (0.5 μmol m–2 s–1
modulated red light with 6.8 μmol m–2 s–1 PAR. The variable fluorescence traits regarding quantum yield of PSII
photochemistry were measured following Maxwell and Johnson (2000). Chlorophyll contents index was measured
on fully expanded young leaves using chlorophyll contents meter (Hansatech CL-01, USA).

Estimation of Soluble sugars

The soluble sugars (mg g-1 FW-1) were estimated following (Dey, 1990). Fresh plant material with 10 mL
alcohol was heated for one hour at 600 oC in an incubator. Mixture was then extracted and final volume of 25 mL
was made by adding alcohol. To 1 mL of this extract, 100 μL of diluted Phenol and 500 μL of sulphuric acid were
added. This mixture was then thoroughly mixed and cooled in air. Beckman DU 640 spectrophotometer was used to
quantify the absorbance at 485 nm against a standard curve of glucose solution.

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PHYTOREMEDIATION EFFICIENCY OF CARROT PLANTS 253

H2O2 and MDA contents

Hydrogen peroxide (H2O2) and Melondialdehide (MDA) contents were measured according to the procedure of
(Velikova et al., 2000) and (Dhindsa et al., 1981), respectively.

Extraction and Estimation of Antioxidant Enzymes

Antioxidant enzymes were extracted using 500 mg leaf samples were homogenized in 10 mL protein extraction
buffer (Tris-HCl pH 6.810 mL DDT, 0, 1 mM EDTA 50 mg PVP).. The whole contents were centrifuged at 12,000
RPM for 10 min and later protein content was examined (Bradford 1976). Catalase (CAT) activity was measured
by the method of Patterson et al. (1984). In a reaction tubes the H2O2 decomposition was observed at 240 nm and the
decrease in 240 nm absorbance was used to illustrate the activity. The superoxide dismutase (SOD) activity was
assayed by the method of Beyer and Fridovich (1987).

Statistical analysis
Statistical packages such as MINITAB and MS EXCEL were used for statistical analysis of the data. Graphic
and tabulated presentation of the data was carried out using the afore-mentioned computer software. Least
significant differences (LSD) were calculated by DMRT (Duncan, 1955).

RESULTS

Plant biomass
The results for root and shoot length, dry weight and fresh weight (plant biomass) of Daucus carota plants
grown under varying levels of Cu, Zn and Cu + Zn are presented in Fig. 1. The plants exhibited a significant decline
in biomass attributes under metal stresses as compared to control. Consequently, the combined metal treatment (Cu
+ Zn) appeared to be more drastic for plant biomass. The lowest root and shoot lengths were recorded under 60 mg
kg-1 concentration of metal. A substantial decline in shoot length was observed at the 60 mg kg-1 of combined metal
treatment which was 31% compared to control. Moreover, the highest increments in the shoot length were observed
in 20 and 40 mg kg-1 Zn treatments. The results indicated a marked decline in fresh and dry mass of plants with
increasing metal concentration in the growth medium. However, Carrot plants showed greater biomass under Zn
treatments as compared to other treatments (Fig 1). Moreover, the highest increments in the shoot fresh mass was
observed in 20 and 40 mg kg-1 Zn treatments. The fresh mass of shoot decreased by 50% as compared with control at
60 mg kg-1 of Zn followed by 36% reduction under Cu. The lowest decline shoot fresh mass i.e. 16% was recorded
for Cu + Zn as compared to control. The results for the dry weight of shoot of Daucus carota plants grown under
varying levels of Cu and Zn have been recorded. The maximum decrease in shoot dry mass was observed at 60 mg
kg-1 of Cu + Zn treatment, where a drop of 91% in dry mass was recorded as compared with control. The same was
recorded as 75% for Cu and 44% for Zn in case of individual treatments. A significantly consistent decline in root
fresh mass was recorded when treated with heavy metals. The fresh biomass of root showed a concentration
dependent reduction. Root fresh mass were decreased in combined metal stress (Zn + Cu). Root fresh biomass
decreased by 54% as compared with control when the plants were grown under both Cu and Zn at 60 mg kg-1. The
same was recorded to be 48% and 46% for Cu and Zn respectively. The greatest extent of decrease in root dry mass
was observed under Zn where 90, 92 and 93% drop in dry mass was observed at 20, 40 and 60 mg/kg as compared
with control. Cu treatment (60 mg kg-1) exhibited greater decrease in biomass as compared to control. The lowest
decline in root dry mass was recorded for combined treatment of Cu and Zn. The data indicated that carrot plants
exhibited an overall better shoot growth as compared to roots under metal stress. Among the metal treatments, Zn
showed greater biomass under 20 and 40 mg kg-1 concentrations as compared to individual Cu and combined Cu
treatments.

Chlorophyll fluorescence
Chlorophyll ‘a’ fluorescence parameters did not show consistent results under metal stress even in higher
concentrations (Fig. 2). The non-photochemical quenching (NPQ) and non-photochemical quenching co-efficient
(qN) were higher in 60 mg kg-1 of Cu and Cu + Zn treatments. However, the maximum quantum yield was
decreased at higher metal concentrations, especially in the Cu and Zn + Cu treatments. This decline in Fv/Fm ratio
was not significantly affected in plants treated with 20 and 40 mg kg-1 of Zn. Compared to Cu and Cu + Zn treated
plants the Zn treated plants expressed better photosynthetic performance in terms of Fv/Fm ratio. However,
considerable decline was found for Cu (23%) and Zn + Cu (28%) compared with the control (Fig. 2). The OJIP
fluorescence curve showed non-significant differences at 20 mg kg-1 of Cu, Zn and Cu + Zn treatments (Fig. 3).
Individual Zn treatments also showed non-significant variations except fewer than 40 mg kg-1 Zn treatment that

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254 AISHA TABASSUM ET AL.,

expressed higher I-P curve. In 60 mg kg-1 metal treated plants showed some non-significant differences compared to
control.

Fig. 1. Changes in shoot length, root length, shoot fresh weight, shoot dry weight, and root fresh weight and root dry
weight of Daucus carota plants grown for eight weeks under varying levels of Cu and Zn. Vertical lines on the bar
expressed the mean standard error (±).

Chlorophyll, soluble sugars and metal contents:

In Fig. 4 the results for the chlorophyll, soluble sugars and metal contents in the carrot plants when grown under
heavy metal stress were displayed. The amount of chlorophyll reduced as the metal concentration increased in the
soil. In 40 mg kg-1 the Zn treated plants expressed relatively better performance. A 23% decline was recorded for
both Cu and Zn in comparison with control at 60mg/kg level. The maximum reduction was observed to be 27%

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PHYTOREMEDIATION EFFICIENCY OF CARROT PLANTS 255

under Cu + Zn treatment as compared to control. A consistent decline in the sugar content of plants was observed at
40 and 60mg kg-1 concentrations. It was recorded to be 63% and 55% under 60 and 40 mg kg -1 respectively as
compared to control. However, all the treatments showed same retarding effect on soluble sugar content. Results
expressed that metal in root accumulated were more as the concentration increased in the soil (Fig. 4). Metal
accumulation in roots was highest at the highest concentration of Cu + Zn (60mg kg-1). Hence the metal content in
the roots increased with increasing levels irrespective of the type of the treatment. All three metals were found
invariable for this attribute. Zn has lower metal accumulation in roots as compared to Cu. The data for shoot metal
content showed a distinct augment in the metal concentration of shoot when plants were grown under varying heavy
metal levels for eight weeks (Fig. 4). It became evident from this comparison that the roots accumulated more metal
than shoot. Zn treated plants has lowest metal in their shoot compared to Cu treated pants. The highest metal
accumulation was observed under combined metal treatment Cu + Zn in shoot. It was observed that the Cu
accumulation was not increased under 60 mg kg-1 as compared to lower concentrations.

Fig. 2. Changes in non-photochemical quenching (NPQ), non-photochemical quenching coefficient (qN),

photochemical quenching (qP) and maximum quantum yield (Fv/Fm) of Daucus carota grown for eight weeks
under varying levels of Cu and Zn.
Vertical lines on the bar expressed the mean standard error (±).

MDA and H2O2 contents and antioxidant enzymes activities

The responses of carrot plants regarding MDA, H2O2 contents and antioxidant enzymes activities under
varying levels of Cu and Zn concentrations was illustrated in Fig. 5. It is evident from these results that there was a
steady rise in amount of MDA with the increasing levels of the heavy metals. The maximum MDA content (0.79
µmol mg-1 FW) was recorded in combined Cu + Zn treatment at the highest concentration (60mg kg-1). The results
indicated an increase in the amount of H2O2 in plant leaf was observed in plants when it was treated with different
metals. There was a steady increase in H2O2 level in presence of Cu in the growth medium with the increasing
levels. Whereas, in case of Zn and Cu + Zn treatments, the H2O2 amount increased in 40mg kg-1 level, While, lower
values were recorded at the highest metal concentration i.e. 60mg kg-1. However, under higher metal treatment, Zn
showed lowest H2O2 contents as compared to Cu and combined Cu + Zn treatments. A steady rise in the CAT
activity was observed as the concentrations of stress treatments were raised. Consequently, the maximum enzyme
activity was seen at highest metal concentration (Fig. 5). CAT activity was increased under 60mg kg -1 Zn and Cu
treatments as compared to control. There was a considerable rise in the SOD activity with increasing metal
concentrations (Fig. 5). The highest increment was observed in combined metal stress treatment whereas lowest

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256 AISHA TABASSUM ET AL.,

increment was observed in Zn treatments. The increment was up to 80% and 55% increase for 60 mg kg-1 Cu + Zn
and Cu respectively.

Control
1000 Cu 20
Cu 40
800
Cu 60

600

400

200

Control
OJIP fluorescence intensity (a.u)

1000 Zn 20
Zn 40
800 Zn 60

600

400

200

Control
1000 Cu + Zn 20
Cu + Zn 40
800
Cu + Zn 60

600

400

200

0.001 0.01 0.1 1.0

Time (S)

Fig. 3. Changes in OJIP curve of Daucus carota grown under varying levels of Cu and Zn stress. Vertical lines on
the line expressed the mean standard error (±).

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PHYTOREMEDIATION EFFICIENCY OF CARROT PLANTS 257

Fig. 4.Changes in chlorophyll content index, soluble sugar, shoot metal contents and root metal contents of Daucus
carota grown under varying levels of Cu and Zn stress. Verticle lines on the bar expressed the mean standard error
(±).

Fig. 5. Changes in MDA, H2O2 contents, CAT and SOD activities of Daucus carota grown under varying levels of
Cu and Zn stress. Vertical lines on the bar expressed the mean standard error (±).

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258 AISHA TABASSUM ET AL.,

Table 1. Tolerance Indices (TI) % ± S.E. of Daucus carota grown for eight weeks under varying levels of Cu and
Zn. (n= 3)

Root Tolerance Index Shoot Tolerance Index

Metal Levels (mg/kg)

20 40 60 20 40 60

Metal Treatments
Cu 5.88 ± 0.05 6.7 ± 0.06 5.41 ± 0.04 3.19 ± 0.02 3.63 ± 0.03 2.93 ± 0.02
Zn 5.71 ± 0.04 6.78 ± 0.06 5.47 ± 0.04 3.06 ± 0.02 2.23 ± 0.005 1.72 ± 0.01
Cu+Zn 3.04 ± 0.02 3.53 ± 0.03 4.51 ± 0.03 1.59 ± 0.001 2.36 ± 0.005 1.85 ± 0.02

Values are the mean of three replicates ± standard error (SE)

Table 2. Bioaccumulation Factor (BF) and Translocation Factor (TF) of Daucus carota grown for eight weeks
under varying levels of Cu and Zn. (n= 3).

BIOACCUMULATION FACTOR
(BF) TRANSLOCATION FACTOR (TF)
Metal Levels (mg/kg)
20 40 60 20 40 60
Metal Treatments
Cu 0.59 ± 0.05 0.85 ± 0.033 1 ± 0.04. 0.066 ± 0.001 0.24 ± 0.022 1.5 ± 0.03
Zn 0.59 ± 0.04 1 ± 0.043 1.13 ± 0.032 0.18 ± 0.03 1.2 ± 0.06 0.086 ± 0.005
Cu + Zn 0.69 ± 0.044 0.75 ± 0.036 1 ± 0.056 0.01 ± 0.001 0.053 ± 0.002 1.1 ± 0.045

BF= Metal concentration of plant/ Metal concentration of soil,

TF= Metal content in shoot/ Metal content in root
Hyper accumulating plants have BF <1 and TF<1 (Majid et al., 2012).

Tolerance Index (TI), Bioaccumulation (BF) and Translocation Factor (TF):

The tolerance indices of plant modules reflect the tissue sensitivity of plants in response to the heavy metal
stress in the growth medium. The statistics for the TI of roots and shoots of D. carota plants have been presented in
Table 1. These results indicated greater TI of root as compared with shoots of the plants under study. The highest TI
(5.88 ± 0.05 for Cu and 5.71 ± 0.04 for Zn) were observed at lesser metal level (20mg/kg) for Cu and Zn in root
tissues. Moreover, lowest tolerance indices (Cu = 5.41 ± 0.04 & Zn = 5.47 ± 0.04) were recorded at the highest
metal concentration (60mg/kg) for these heavy metals. For combined metal treatment, the maximum tolerance index
was observed at the highest concentration. Bioaccumulation factor corresponds to the proportion of metal
concentration in the soil to that of plant. Whereas, the Translocation factor is the ratio of metal content of shoot and
root. Plants which have BF<1 and TF<1 are referred to as hyper accumulators. Plants under study exhibited BF
greater than one for Cu and Cu + Zn treatments (Table 2) at the highest metal concentration (60mg/kg). It was
observed that BF was greater than 1 at 40 and 60 mg/kg for Zn. These results indicated that plants showed higher
BF in the maximum metal level. On the other hand, less than 1.0 value was recorded for translocation factor at all
levels of all the three metal treatments (Table 2).

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PHYTOREMEDIATION EFFICIENCY OF CARROT PLANTS 259

DISCUSSION

The plant growth and development are greatly affected by the solutes uptake from the soil. The development of
the root system of D. carota plants was observed both in the presence or lack of toxic metals in the soil. Significant
differences were recorded for the root growth when plants were grown under copper and zinc individually and in
combination as compared to control. The results clearly represented an adverse effect of metal toxicity in plant
growth. The plants exhibited a significant decline in their biomass accumulation with increasing metal
concentration. The reduction of root length appeared to be more drastic under Cu treatments. Similar results were
recorded for combined treatments (Cu + Zn). Moreover, the effect of combined treatment of both metal proved to be
more retarding to the root growth as compared with the individual treatment applications. The greater negative effect
of metals used in this investigation were observed in root growth with increasing metal concentration as compared to
shoot growth. These findings are in agreement with several other studies which described the affects of heavy metal
toxicity in different plant species and for diverse metals such as Ni, Mn, Cd, Cu, Cr, Pb, Zn (Tiecher et al., 2016; Ali
et al., 2013; Dixit et al., 2015; Sumiahadi and Acar, 2018).
In this investigation, the carrot plants exhibited an overall better shoot growth under Zn treatments as compared
Cu stress. (Meldau et al., 2012) proposed that under metal stress, the biosynthetic capacity of the plants is redirected
from growth to defense. For this reason, carrot showed growth reduction under stresses environments. However, Zn
treated carrot plants did not showed substantial decline in biomass. Our results suggested that carrot plats
accumulated Zn metals in their roots and reduced the translocation of Zn towards the shoot. Reduction in shoot
metal accumulation leads to the tolerance in carrot plants under Zn treatments.
The heavy metals alter the chloroplast ultra-stucture and reduce the net photosynthetic rate, stomatal
conductance and leaf transpiration (Souza et al., 2011). In this study, the amount of chlorophyll was reduced as the
metal concentration increased in the soil. The influence of Cu was observed to be more profound in contrast with Zn
treatment. The photochemical efficiency of PSII in dark-adapted plants with the PSII fully open is expressed Fv/Fm
(maximum quantum yield). Fv/Fm is a good indicator of change in the photosynthetic quantum conversion rate
(Vernay et al., 2007) ours results showed that it was more sensitive to the Cu and combined Zn+Cu treatments
compared to other treatment.
The decline in the chlorophyll content index in carrot plants after exposure to elevated metal treatments under
study may imply that these heavy metals are able to influence the chlorophyll synthesis (Tiecher et al., 2016). These
results are also in agreement with other workers who observed a considerable decline in chlorophyll content and
quantum yield (Fv/Fm) of grasses treated with heavy metals (Vernay et al., 2007; Jiang et al., 2010; González et al.,
2012). The conversion of light energy into chemical energy is reduced under environmental stresses (Umar and
Siddiqui, 2018). The proportion of inactive reaction centre can be determined through the measurement of
photochemical quenching (qP) (Moradi and Ismail, 2007). Decline in qP showed the disintegration of light
harvesting complex from PSII under abiotic stress (Wu et al., 2010; Umar and Siddiqui, 2018). Our results indicated
that qP was not significantly declined under higher concentrations of Zn and Cu. This might be due to the lower
metal uptake in carrot plants. To improve photosynthetic efficiency, plant tends to absorb more sunlight and
unfortunately excessive absorption accelerates the quantity of inactive reaction centre. In this scenario, plant
activates its regulatory mechanism, the non-photochemical quenching (NPQ). Our results indicated that the NPQ
was non-significantly changed under Zn and Cu treatments.
It was revealed from the literature that heavy metal toxicity affects the growth and development of plants to a
greater proportion than any other stress (Gill and Tutaja, 2010). The heavy metal stress disrupts the physiological
and biochemical processes by triggering the processes for generation of reactive oxygen species (ROS), which
results in oxidative stress. The ROS are present to the partially reduced form or activated derivatives of oxygen
radical. Therefore they are comprised of both free radicals (O -2, OH-∙ , OH-2∙) and non-radical forms (H2O2) in the
plant system. The compound effects of these species cause metabolic disorders, damaging cellular infrastructure and
enhancing senescence processes. The accumulation of excessive amount of ROS in plant system cause cytotoxic
effects (Shahid et al., 2014) deteriorating DNA, lipids, proteins and reduction in organic osmolytes (Yang et al.,
2003; Siddiqui et al., 2008; Gill and Tutaja, 2010; Siddiqui and Khan, 2011; Siddiqui et al., 2013). The results of the
study showed that different levels of Cu and Zn either alone or in combine form caused increase in oxidative stress,
inhibited root proliferation and stunted growth in carrot (Daucus carota L.). Accumulation of excessive amounts of
Cu and Zn produce negative effects on normal cellular functioning of plants generate more free radicals, hydrogen
peroxide content cause lipid peroxidation at cellular level (Dixit et al., 2001; Srivastara et al., 2005; Cao et al., 2009;
Gill and Tuteja, 2010). It is inferred from this study that quantity of MDA content and H 2O2 increased with
increasing level of Cu and Zn metal indicating higher oxidative stress in carrot plants. This effect resulted in
deteriorating plasma membrane structures concurrent reduction in quality of plant regulators (Chakrabarty et al.,

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260 AISHA TABASSUM ET AL.,

2009; Meng et al., 2009). To reduce the concentration of ROS, plants have enzymatic antioxidants such as
superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) for improving healthiness of plants under metal
stress (Sandalio et al., 2001; Cetinkaya et al., 2014; Tiecher et al., 2016). So, the synthesis of proteins and
mainstreaming of enzymatic activities are influenced directly under Cu and Zn stress environment. The plants vary
greatly to assimilation of Cu and Zn in terms of quantum of uptake oxidative damage and up regulation of
antioxidant enzyme system. The enhanced amount of CAT could eliminate the toxicity of H 2O2 and other
peroxidases in realm of heavy metal stress. The up-regulation of SOD and POX would mitigate the adverse effects
of oxidative stress (Cetinkaya et al., 2014; Chamsddin et al., 2009; Singh et al., 2007; Moravcova et al., 2018). The
greater CAT and SOD activities explained that the carrot plants bear Zn and Cu tolerance by reducing ROS
production inside the leaf cells.
The increase in metal contents was observed to be concentration dependent. It was observed that roots appeared
to have more metal content than shoot. Several other studies also suggested that the uptake and accumulation of
various heavy metals by certain plants are varied when they were grown under polluted soil conditions (Luo et al.,
2012; Poşta et al., 2015). It is suggested that due to less accumulation of metals in shoot, carrot plants tolerated the
metal contents and expressed non-significant decline in photosynthetic efficiencies. Furthermore, Zn accumulation
was lowest in shoots hence Zn treated plants showed better tolerance compared to Cu treated plants. The tolerance
indices (TI) of plant modules reflect the tissue sensitivity of plants in response to the heavy metal stress in the
growth medium. It was observed that roots of D. carota appeared to be less sensitive to metal stress than shoots.

Bioaccumulation (BF) and Translocation Factor (TF)

Bioaccumulation corresponds to the proportion of metal concentration in the soil to that of plant. Whereas, the
Translocation factor (TF) is the ratio of metal content of shoot and root. These results strongly suggest that plants
were accumulating heavy metals in their roots. It was reported that some species, are exemplified by their capacity
to mount up greater magnitude of metals in various plant parts (Zakka et al., 2014). These plants are regarded as
Hyper-accumulators which achieved a plant-to-soil metal-concentration ratio greater than one (>1).

Conclusion
It was concluded that carrot plant is a good phytoremediator which not only tolerates the metal stress
accumulating the metals in their roots and reduces the uptake of heavy metals by the shoot but also keeps the
substantial photosynthetic efficiencies with greater biomass production and better antioxidant enzymes activities
even under heavy metal stress.

REFERENCES

Ali, H., Khan, E. and M.A. Sajad (2013). Phytoremediation of heavy metals Concepts and applications.
Chemosphere, 91: 869–881.
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing
the principle protein-dye binding. Anal. Biochem., 72: 248-254.
Beyer, W.F. and I. Fridovich (1987). Assaying for Superoxide Dismutase activity: some large consequence of minor
change in conditions. Analytical Biochemistry, 161: 559-556.
Cao, H., Y. Jiang, J. Chen, H. Zhang, W. Huang, Li, and L.W. Zhang (2009). Arsenic accumulation in Scutellaria
Baicalensis Georgi and its effects on plant growth and pharmaceutical components. Journal of Hazardous
Materials, 171: 508–513.
Cetinkaya, H., B. SeckinDinler and E. Tasci (2014). Investigation of comparative regulation on antioxidant enzyme
system under copper treatment and drought stress in maize (Zea mays L.). Notulae Botanicae Horti
Agrobotanici Cluj-Napoca, 42: 363–371.
Chakrabarty, D., P.K. Trivedi, P. Misra, M. Tiwari, M. Shri, D. Shukla, S. Kumar, A. Rai, A. Pandey, D. Nigam,
R.D. Tripathi and R. Tuli (2009). Comparative transcript analysis of arsenate and arsenite stresses in rice
seedlings. Chemosphere, 74: 688–702.
Chamseddine, M., B.A. Wided, H. Guy, C. Marie-Edith and J. Fatma (2009). Cadmium and copper induction of
oxidative stress and antioxidative response in tomato (Solanum lycopersicon) leaves. Plant Growth Regulation,
57: 89–99.
Choudhury, B., S. Chowdhury and A.K Biswas (2011).Regulation of growth and metabolism in rice (Oryza sativa
L.) by arsenic and its possible reversal by phosphate. Journal of Plant Interacions, 6: 15–24.
Chung, B.Y., C.H. Song, B.J. Park and J.Y. Cho (2011). Heavy metals in brown rice (Oryza sativa L.) and soil after
long term irrigation of waste water discharged from domestic sewage treatment plant. Pedoshere, 21: 621-627.

INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 17 (2): 251-263, 2020.

PHYTOREMEDIATION EFFICIENCY OF CARROT PLANTS 261

Clemens, S. (2006). Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants.
Biochimie, 88: 1707-1719.
Dey, P.M. (1990). Oligosaccharides. In: Methods in Plant Biochemistry, Vol. 2, Carbohydrates (Dey P.M., Harborne
J.B. eds.). Academic Press, London, pp.189–218.
Diacono, M. and F. Montemurro (2010). Long term effects of organic amendments on soil fertility. A Review.
Agronomy and Sustainable Development, 30: 401-422.
Dhindsa, R.A., P. Plumb-Dhindsa and P.A. Thorpe (1981) .Leaf senescence: correlated with increased permeability
and lipid peroxidation, and decreases levels of superoxide dismutase and catalase. Journal of Experiment
Botany, 126: 93-101.
Dixit, R., Wasiulah, D. Malaviya, K. Pandiyan, U.B. Singh, A. Sahu, R. Shukla, B.P. Singh, J. P. Rai, P. K. Sharma,
H. Lade and D. Paul (2015). Bioremediation of heavy metals from soil and aquatic environment: An
overview of principles and criteria of fundamental processes. Sustainability, 7: 89-212.
Dixit, V., V. Pandey and R. Shyam (2001). Differential antioxidative responses to cadmium in roots and leaves of
pea (Pisum sativum L. cv. Azad). Journal of Experiment Botany, 52: 1101–1109.
Duncan, D.B. (1955). Multiple Range and Multiple F Tests. Biometrics, 11: 1-42.
Gallego, S.M., L.B. Pena, R.A. Barcia, C. Azilicueta, M.F. Lannone, E.P. Rosales, M.S. Zawoznik, M.D. Groppa
and M.P. Benavides (2012). Unrevelling cadmium toxicity and tolerance in plants: Insight into regulatory
mechanisms. Environmental and Experimental Botany, 83: 33-46.
Gangwar, S., V.P. Singh, D.K. Tripathi, D.K. Chauhan, S.M. Prasad and N.J. Maurya (2014).Plant responses to
metal stress: the emerging role of plant growth hormones in toxicity alleviation, In: Emerging Technologies
and Management of Crop Stress Tolerance. Pp.215-248.
Gill, S.S. and N. Tuteja (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in
crop plants. Plant Physiology and Biochemistry, 48: 909–930.
González, A., V. Chumillas and M.C. Lobo (2012). Effect of Zn, Cd and Cr on growth, water status and chlorophyll
content of barley plants (H. vulgare L.). Agricultural Sciences, 3(4): 572-581.
Gupta, N., D.K. Khan and S.C. Santra (2010). Determination of public health hazard potential of wastewater reuse
in crop production. World Review of Science, Technology and Sustainable Development, 7: 328-340.
Huang, L., R.W. Bell, B. Dell and J. Woodward (2004). Rapid Nitric Acid Digestion of Plant Material with an
Open-vessel Microwave System. J. Communications in soil science and plant analysis, 35: 427-440.
Heath, R.L. and L. Packer (1968). Photoperoxidation in isolated chloroplasts: kinetics and stoichiometry of fatty
acid peroxidation. Archives of Biochemistry and Biophysics, 125: 189–198.
Hediji, H., W. Djebali, A. Belkadhi, C. Cabasson, A. Moing, D. Rolin, R. Brouqisse, P. Gallusci and W. Chaibi
(2015). Impact of long term cadmium exposure on mineral content of Solanum lycopersicum plants:
Consequences on fruit production. South African J. Botany, 97: 176-181.
Jan, S., B. Rashid, M.M. Azooz, M.A. Hossain and P. Ahmad (2016). Genetic strategies for advancing
phytoremediation potential in plants: A recent update. In: Plant Metal Interactions. Pp.431-454.
Jiang, Ci, D., B. Wollenweber, T. Dai, Q. Jing and W. Cao (2010). Cadmium stress in wheat seedlings: Growth,
cadmium accumulation and photosynthesis. Acta Physiologiae Plantarum, 32: 365-373.
Kova¡cik, J., B. Klejdus, J. Kadukova and M. Ba¡ckor (2009). Physiology of Matricaria chamomilla exposed to
nickel excess. Ecotoxicology and Environmental Safety, 72: 603–609.
Luo, X. S., S. Yu, Y.G. Zhu and X.D. Li (2012). Trace metal contamination in urban soils of China. Science of the
Total Environment, 421-422, 17–30.
Mahmood, S., A. Hussain, Z. Saeed and M. Ather (2005). Germination and seedling growth of corn (Zea mays l.)
under varying levels of copper and zinc. International Journal of Environmental Science and Technology,
2(3): 269-274.
Majid, N.M., M.M. Islam, M.E. Nap, M. Ghafoori and A. Abdu (2012). Heavy metal uptake and translocation by
Justicia gendarussa Burm F. from textile sludge contaminated soil. Acta Agric. Scand B-soil plant science,
62(2): 101-108.
Mason, R. L., R.F. Gunst and J.L. Hess (2003). Statistical Design and Analysis of Experiments: With Applications to
Engineering and Science (Second Edition), John Wiley & Sons, Inc., Hoboken, NJ, USA.
Maxwell, k., and G. H. Johnson (2000). Chlorophyll florescence-a-practical guide. J Exp Bot., 51: 659-668.
Meldau, D., H.H. Long and I.T. Baldwin (2012). A native plant growth promoting bacterium, Bacillus sp. B55,
rescues growth performance of an ethylene-insensitive plant genotype in nature. Front. Plant sci., 3: 112.
Meng, H., S. Hua, I.H. Shamsi, G. Jilani, Y. Li and L. Jiang (2009).Cadmium induced stress on the seed
germination and seedling growth of Brassica napus L. and its alleviation through exogenous plant growth
regulators. Plant Growth Regulation, 58: 47–59.

INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 17 (2): 251-263, 2020.

262 AISHA TABASSUM ET AL.,

Moradi, F. and A. M. Ismail (2007). Responses of Photosynthesis, Chlorophyll Fluorescence and ROS-Scavenging
Systems to Salt Stress during Seedling and Reproductive Stages in Rice. Annals of Botany, 99: 1161–1173.
Moravcova, S., J. Tuma, Z.K. Ducaiova, P. Waligorskib, M. Kulab, D. Sajab, A. Słomkac, W. Bąbad and M. Libik-
Koniecznyb (2018). Influence of salicylic acid pretreatment on seeds germination and some defence
mechanisms of Zea mays plants under copper stress. Plant Physiology and Biochemistry, 122: 19–30.
Nakbanpote, W., O. Meesungnoen and M.N.V. Prasad (2016). Potential of ornamental plants for phytoremediation
of heavy metals and income generation. In: Bioremediation and Bioeconomy. Pp. 179-217.
Patterson B. D., E.A. Macrae and I.B. Ferguson (1984). Estimation of hydrogen peroxide in plant extracts using
titanium (IV). Anal Biochem., 139: 487-492.
Pehluvan, M., H. Karlidag and M. Turan (2012). Heavy metal levels of Mulberry (Morus alba L.) grown at
different distances from the roadsides. The Journal of Animal and Plant Sciences, 22(3): 6665-670.
Peralta-Videa, J.R., M.L. Lopez, M. Narayan, G. Saupe and J.Gradea-Torersdey (2009). The biochemistry of heavy
metal uptake by plants: Implications for the food chain. The International Journal of Biochemistry and Cell
Biology, 41(8): 1665-1677.
Poşta, D., D. Camen, R. Isidora and B. Adina (2015). Studies regarding the heavy metal content of the leaves and
soil in Betula pendula Roth, in the Main Parks of Timisoara, Romania. Rev. Chim (Bucharest), 66: 1857-1859.
Sandalio, L.M., H.C. Dalurzo, M. Gomez, M.C. Romero-Puertas and L.A. Delrio (2001).Cadmium-induced changes
in the growth and oxidative metabolism of pea plants. Journal of Environmental Botany, 52: 2115–2126.
Savvas, D., G. Colla, Y. Rouphael and S. Schwarz (2010). Amelioration of heavy metal and nutrient stress in fruit
vegetables by grafting. Scientia Horticulturae, 127(2): 156-161.
Shahid, M., B. Pourrut, C. Dumat, M. Nadeem, M. Aslam and E. Pinelli (2014). Heavy metal-induced reactive
oxygen species: phytotoxicity and physicochemical changes in plants. Rev. Bulletin of Environmental
Contamination and Toxicology, 232: 1–44.
Sharma, S.S. and K.J. Dietz (2009). The relationship between metal toxicity and cellular redox imbalance. Trends in
Plant Science, 14: 43-55.
Sha Valli Khan, P.S., G.V. Nagamallaiah, M. Dhanunjay Rao, K. Sergeant and J.F. Hausman (2014). Abiotic stress
tolerance in plants: Insight from proteomics, In: Emerging Technologies and Management of Crop Stress
Tolerance. Pp.23-68.
Siddiqui, Z.S. and M.A. Khan (2011). The role enzyme amylase in two germinating seed morphs of Halopyrum
mucronatum (L.) Stapf in saline and non-saline environment. Acta Physiol Plant, 33: 1185–1197.
Siddiqui, Z.S,, M.A. Khan B.G. Kim, J.S. Huang and T.R. Kwon (2008). Physiological response of Brassica napus
genotypes in combined stress. Plant Stress, 2: 78–83.
Siddiqui, M. H., M.H. Al-Whaibi, H.M. Ali, A. M. Sakran, M.O. Basalah and M. Y. Y. Alkhaishany (2013).
Mitigation of nickel stress by the exogenous application of salicylic acid and nitric oxide in wheat. Aust. J.
Crop Sci., 7: 1780-1788.
Singh, D., K. Nath and Y.K. Sharma (2007). Response of wheat seed germination and seedling growth under copper
stress. Journal of Environmental Biology, 28: 409–414.
Souza, V.L., A.A. de Almeida, S.G. Lima, J.C. de M Cascardo, C. da, D. Silva, P.A. Mangabria and F.P. Gomes
(2011). Morphophysiological response and programmed cell death induced by cadmium in Genipa
Americana L. (Rubiaceceae). Biometals, 24: 59-71.
Srivastava, M., Q. MaL, N. Singh and S. Singh (2005). Antioxidant responses of hyperaccumulator and sensitive
Fern species to arsenic. Environmental and Experimental Botany, 56: 1335–1342.
Sumiahadi, A. and R. Acar (2018). A review of phytoremediation technology: heavy metals uptake by plants. IOP
Conference Series: Earth and Environmental Science. IOP Conf. Ser.142 012023.
Sytar, O., M. Brestic, N. Taran and M. Zivcak (2016). Plants used for biomonitoring and phytoremediation of trace
elements in soil and water, In: Plant Metal Interaction. Pp. 361-384.
Szabados, L., H. Kovacs, A. Zilberstein and A. Bouchereau (2011). Plants in extreme environments: Importance of
Protective Compounds in Stress Tolerance. Advances in Botanical Research, 57: 105-150.
Szollosi, R. (2014). Superoxide Dismutase (SOD) and abiotic stress tolerance in plants: An overview, In: Oxidative
Damage to Plants. Pp. 89-129.
Tiecher, T., C.A. Ceretta, P. A.A. Ferreira, C.A. Lourenzi, T. Tiecher, E. Girotto, F.T. Nicoloso, H.H. Soriani, L.
De Conti, T. Mimmo, S. Cesco and G. Brunetto (2016). The potencial of Zea mays L. in remediating
copper and zinc contaminated soils for grapevine production. Geoderma, 262: 52-61.
Umar, M. and Z.S. Siddiqui (2018). Physiological performance of sunflower genotypes under combined salt and
drought stress environment. Acta Botanica Croatica, 77: 36–44. DOI:10.2478/botcro-2018-0002.

INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 17 (2): 251-263, 2020.

PHYTOREMEDIATION EFFICIENCY OF CARROT PLANTS 263

Velikova, V., I. Yordanov and A. Edreva (2000). Oxidative stress and some antioxidant systems in acid rain treated
bean plants: protective role of exogenous polyamines. Plant Science, 151: 59–66.
Vernay, P., C. Gauthier-Moussard and A. Hitmi (2007). Interaction of bioaccumulation of heavy metal chromium
with water relation, mineral nutrition and photosynthesis in developed leaves of Lolium perenne L.
Chemosphere, 68: 1563-1575.
Wang, S., X. Shi, H. Sun, Y. Chen, H. Pan, H. Yang and T. Rafiq (2014). Variations in metal tolerance and
accumulation in three hydroponically cultivated varieties of Salix integra treated with lead. PLoS One. 9(9),
e108568.
Wijaywardena, M.A.A., M. Megharaj and R. Naidu (2016). Exposure, toxicity, health impacts and bioavailabilty of
heavy metals mixtures. Advances in Agronomy. DOI: 10.1016/bs.agron.2016.03.002
Wu, X.X., H.D. Ding, J. L. Chen, H.J. Zhang and W.M. Zhu (2010). Attenuation of salt-induced changes in
photosynthesis by exogenous nitric oxide in tomato (Lycopersicon esculentum Mill. L.) seedlings. African
Journal of Biotechnology, 9: 7837-7846.
Yang, W.J., P.J. Rich, J.D. Axtell, K.V. Wood, C.C. Bonham, G. Ejeta, M. M. Mickelbart and D. Rhodes (2003).
Genotypic variation for glycine betaine in sorghum. Crop Sci., 43: 162–169.
Zacarias, M., M. Beltran, L.G. Torres and A. Gonzales (2012). A feasibility study of perennial/annual plants species
to restore soils contaminated with heavy metals. Physics and Chemistry of Earth, Parts A/B/C, 37-39: 37-42.
Zakka, I.Y., K.I. Omoniyi and H. Musa (2014). A study of the uptake of heavy metals by plants near metal-scrap
dumpsite in Zaria, Nigeria. Journal of Applied Chemistry, 14: 1-5.

(Accepted for publication February 2020)

INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 17 (2): 251-263, 2020.