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Research Paper
Phytoremediation potential of hemp (Cannabis sativa L.): Identification and
characterization of heavy metals responsive genes†
Rafiq Ahmad1, Zara Tehsin1, Samina Tanvir Malik2, Saeed Ahmad Asad3, Muhammad Shahzad1, Muhammad
Bilal1, Mohammad Maroof Shah1 and Sabaz Ali Khan1,*
1Department of Environmental Sciences, COMSATS Institute of Information Technology, 22060, Abbottabad,
Pakistan
2Department of Botany, University of Agriculture, Faisalabad, Pakistan
3Center for Climate Research and Development, COMSATS University, Chak Shahzad, Islamabad, Pakistan
Correspondence: Dr. S. A. Khan, Department of Environmental Sciences, COMSATS Institute of Information
Technology, 22060, Abbottabad, Pakistan
Email: sabaz@ciit.net.pk, sabzktk@yahoo.com
†This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi:
[10.1002/clen.201500117]
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: 6 March 2015; Revised: 6 March 2015; Accepted: 15 June 2015
1
Abstract
Soil pollution caused by heavy metals is one of the major problems throughout the world. To maintain a safe and
healthy environment for human beings, there is a dire need to identify hyperaccumulator plants and the
underlying genes involved in heavy metals stress tolerance and accumulation. The goal of this research is to
explore the potential of hemp as a decontaminator of heavy metals by identifying the two important heavy metals
responsive genes, glutathione-disulfidereductase (GSR) and phospholipase D-α (PLDα). The results revealed
heavy metals accumulation; Cu (1530 mg kg--1), Cd (151 mg kg--1) and Ni (123 mg kg--1) in hemp plants’ leaves
collected from the contaminated site. This shows the ability of the hemp plant to tolerate heavy metals, perhaps
due to the presence of stress tolerance genes. In this study, partial sequences of putative GSR (215 bp) and PLDα
(517 bp) genes were identified, responsive to heavy metals stress in hemp leaves. Both genes exhibited 40--60%
sequence identity to previously reported genes from other plant species. Glutathione binding residues and
conserved arginine residues were found identical in a putative GSR gene to those of other plant species, while the
phospholipids binding domain and catalytic domain were found in the PLDα gene. These results will help to
improve our understanding about the phytoremediation potential of hemp as well as in manipulating GSR and
PLDα genes in breeding programs to produce transgenic heavy metals tolerant varieties.
Keywords: Gene characterization, Gene identification, Hyperaccumulator, Glutathione reductase gene,
Phospholipase gene
Abbreviations: GSR, glutathione-disulfide reductase; PA, phosphatidic acid; PIP5K, phosphatidylinositol-4-
phosphate 5-kinase; PLDα, phospholipase D-α; ROS, reactive oxygen species; RT-PCR, reverse transcriptase-
polymerase chain reaction
1 Introduction
Soil contamination is increasing at an alarming rate due to a number of human activities such as release of
industrial effluents, municipal wastes and waste sludge enriched with heavy metals that contaminate the
surrounding environment [1--3]. Once the heavy metals contaminate the environment, they will remain a
potential threat to humans and animals for many years [4]. Biological decontamination methods are considered
safe for removing these pollutants, particularly from water and soil. In this regard, the phytoremediation
technology is an environment friendly and cost-effective technology [5]. Many plant species have the ability to
grow on contaminated sites and some of them are able to accumulate high concentrations of heavy metals in
their tissues. To promote phytoremediation, it is necessary to find hyperaccumulater plants that have the ability
to grow fast and accumulate high concentrations of metals [6]. Currently, more than 400 species of metal
hyperaccumulator plants have been reported in the literature [7]. However, their low roots and shoots biomass
1
strongly limits their real application in this soil decontamination approach. An ideal plant for phytoremediation
should have high biomass, a high tolerance to heavy metals stress and a high metals accumulating capability [8].
Hemp (Cannabis sativa L.) is an annual herb used in many types of non-food industries e.g. it provides raw
material for natural fiber production [9]. There are certain characteristics of hemp, which make it very suitable
for phytoremediation such as high biomass, long roots and a short life cycle of 180 days. In addition, hemp has a
very high capability to absorb and accumulate heavy metals like lead, nickel, cadmium, zinc and chromium [9--
11].
Identification of the functional genes or proteins that are involved in response to heavy metals stress is a
fundamental step in understanding the molecular mechanisms of stress responses. A variety of reactive oxygen
species (ROS) are produced in plants under biotic and abiotic stress conditions. ROS cause severe oxidative
damage to biological molecules, DNA, proteins and lipids. Cells and tissues protect themselves from oxidative
damage through up-regulation of a wide variety of antioxidant products [12, 13]. Among antioxidants,
glutathione-disulfide reductase (GSR) is an abundant metabolite in plants with its diverse and important
functions in the ascorbate glutathione cycle and signal transduction [14, 15]. Recent research has documented a
regulatory role for glutathione in influencing the expression of many genes which are important in plants'
responses to both biotic and abiotic stresses [16]. GSR enzyme activity has been studied in response to different
environmental stresses such as salinity, heavy metals and it protects cells from oxidative damage [16]. Another
class of antioxidant enzymes is the phospholipases. Phospholipases are key enzymes that catalyze the hydrolysis
of structural phospholipids to form its product phosphatidic acid (PA) [17]. PA acts as a secondary messenger
that regulates different protein like kinases, small G proteins, and PIP5K [18--22]. The phospholipase D-α (PLD)
gene and its product PA are involved not only in stress signaling, but also in plant developmental signaling. The
PLD gene has been suggested to regulate specific developmental and stress responses [23, 24]. So far, multiple
forms of phospholipases such as D, C, and A have been characterized in plants. These enzymes are involved in
cellular regulation, lipid metabolism, and membrane remodeling [25]. PLD genes play an important role in the
signaling and production of hormones during different stress responses in plants [25, 26]. The transcription
activity of both PLD and GSR genes increases upon exposure to various stresses, such as cold, drought and
salinity [27--30].
The purpose of the present research is to identify the potential of hemp plants to decontaminate heavy metals
polluted soils. This was done first by summarizing the already published data. Secondly, we identified and
characterized GSR and PLDα genes from hemp plants. The hemp plant genome is still not sequenced and the
genes responsible for heavy metals stress tolerance are unknown. Therefore, the identification, isolation and
characterization of GSR and PLDα genes may allow us to deduce molecular pathways involved in metals
tolerance and accumulation in the hemp plant.
2 Materials and methods
2.1 Samples collection and heavy metals determination
Hemp plant samples were collected from a metals contaminated site near Kohi Noor Textile mills in Rawalpindi,
1
Pakistan. The metals discharged from this industry include Pb, Zn, Cu, Co, Ni, Cr and Cd. Leaves of hemp
plants were harvested and immediately collected in liquid nitrogen and preserved at --80 °C until further
processing. For heavy metals determination, plant leaves were rinsed with distilled water and dried to a constant
weight in an oven at 70 °C. The dried samples were ground, using an agate pestle mortar and kept in polythene
bags. 2 g of prepared soil sample was digested with 15 mL nitric acid, 20 mL perchloric acid and 15 mL
hydrofluoric acid and heated for 3h. After cooling, the digest was filtered into 100 mL flasks and diluted with
distilled water. Likewise, dry powdered leaf samples were digested with 60% HClO4, concentrated HNO3 and
H2SO4. The digested samples were analyzed for heavy metals (As, Cd, Co, Cu, Fe, Ni, Pb and Zn) using atomic
absorption spectrophotometry in the COMSATS Institute of Information Technology, Abbottabad, Pakistan. The
instrument settings and operational conditions were done in accordance with the manufacturers’ specifications.
2.2 Nucleic acids (RNA) isolation
Total RNA extraction was carried out using the RNA prep plant pure RNA extraction kit (TIANGEN Biotech,
Korea). To remove co-extracted DNA, the RNA samples were treated with DNaseI (TIANGEN Biotech, Korea).
Total RNA concentration was determined by spectrophotometry at A = 260 nm, Eq. (1):
(1)
The quality of total RNA was checked by running RNA samples on 1.5% agarose gel.
2.3 Reverse transcriptase-polymerase chain reaction (RT-PCR)
To perform RT-PCR, cDNA was prepared from 2 µg of DNaseI-treated RNA samples, using oligo (dT) primer
and reverse transcriptase enzyme (Topscript cDNA synthesis kit, Enzynomics, Seoul). RT reactions were
incubated at 37 °C for 60 min and then heated at 94 °C for 10 min. Fragments of GSR and PLDα genes were
PCR-amplified from 50 to 100 ng of cDNA equivalent, using degenerated primers. For the amplification of the
PLDα gene, forward primer 5'-CACCATGATGATTTTCATCAGCC-3' and reverse primer, 5'-
TATCATCAACAATCAT-3' and for the GSR gene forward primer 5'-GGAGCATCTTATGGAGGTGAAC-3'
and reverse primer 5'-CAGTTTTTTCTTGTCGCCCAG-3' were used. All oligonucleotide primers were obtained
from Generay Biotech (Shanghai). The total volume used for PCR reaction was 20 μL, containing 50 ng cDNA,
5 pmol of each pair of primers and 10 µL of 2X Master Mix (Taq DNA polymerase, dNTPs, MgCl2 and reaction
buffers). The thermocycler “Master cycler gradient” (Applied Biosystem) was used for amplification of the
genes of interest. The reaction mixture was subjected to the following amplification program: Preliminary
denaturation (94°C, 5 min), followed by 35 cycles of amplification (denaturation; 94°C, 30 s), hybridization
60°C, 30 s), elongation (72°C, 0.45 s) and final elongation at 72°C for 10 min.
2.4 Analysis of PCR products (amplicons)
The DNA fragments amplified by PCR were visualized on 1.5% agarose (Tiangen Biotech, Shanghai) gel
electrophoresis in TAE buffer (0.04 M Tris, 0.001 M EDTA, pH 8) (Gendepot, USA), containing 0.60 µg mL--1
ethidium bromide. A DNA ladder of 100bp-3kb (Enzynomics, Seoul, Korea) was used to compare the fragment
sizes.
2.5 Sequence analysis
The PCR product obtained was sent to Laragen sequencing and genotyping (Virginia, CA). Nucleotide sequence
1
identity was determined using the online BLASTn program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) while
multiple sequence alignments were constructed using the online ClustalW2 program.
3 Results and Discussion
3.1 Hemp potential as a hyperaccumulator
The discovery of hemp’s immense high potential in soil restoration began in 1998 in Ukraine’s Institute of Bast
Crops, where hemps were exclusively planted for the purpose of removing contaminants near the Chernobyl site.
A number of studies conducted on hemp (Cannabis sativa L.) revealed that it can accumulate a considerable
amount of heavy metals from contaminated soils due to its high biomass and deep roots, making it a good
candidate for soil remediation. A hyperaccumulator plant is one that absorbs toxins, such as heavy metals, to a
greater concentration than that the soil in which it is growing [31]. The results showed a higher accumulation of
Cu (1530 mg kg--1), Cd (151 mg kg--1) and Ni (123 mg kg--1) in hemp leaves collected from heavy metals
contaminated sites, whereas all other heavy metals concentration remained negligible (Table 1). Therefore, the
results suggest that hemp plants could be used for the remediation of Cu, Cd and Ni contaminated soils. Many
studies have reported the accumulation of a variety of heavy metals such as Ni, Pb, Cd, Zn and Cr in hemp
(Table 1), makes it suitable to be considered in soil remediation processes. In addition, a number of different
studies have shown that heavy metals accumulation in hemp plants increases with the increasing concentration of
metals in the growing solution/soil. However, the metal distribution in various parts of hemp is different. It has
been proved that a higher concentration of metals in soils increases the heavy metals transfer from roots to hemp
leaves and shoots. Moreover, accumulation tendencies of metals like cadmium, copper, and zinc in hemp are
very similar to each other [32]. With the highest concentration of Cu, greater accumulation in both shoots and
roots of hemp was observed. Similarly, when hemp was treated with 150 mg/kg Cu, a two- and eightfold
increase in the Cu concentration could be observed in shoots and roots, respectively, as compared to the control
[33]. Pb, Zn and Cd phytoextraction potential was studied in hemp with and without the addition of a chelate (10
mmol/kg EDDS). Pb, Zn and Cd concentrations in the biomass of Cannabis sativa were recorded as 1053 ± 125,
211 ± 16 and 5.4 ± 0.8 mg/kg, respectively. It was calculated that the ratios were 105, 2.3 and 31.7 times higher
for Pb, Zn, and Cd, respectively, than in the control experiments. Similarly, Shi and Cai studied the Cd
accumulation and tolerance in eight potential energy crops and they found that hemp is the best Cd accumulator
and is considered an excellent candidate for phytoremediation [34]. In the same way, Linger and his colleagues
conducted experiments to study the growth and photosynthesis ability of hemp under different cadmium
concentrations. They observed that hemp could maintain its growth as well as the photosynthetic machinery, and
long-term acclimation to constant Cd stress [35]. Shi and Cai observed that hemp has a strong tolerance for high
Zn concentrations and showed small inhibitions in plant growth and photosynthetic activities [36]. In another
study, hemp was grown and tested in two different soils, S1 and S2, containing 27, 74, 126 and 82, 115, 139 μg
g−1 of Cd, Ni and Cr, respectively. The results revealed that the mean shoot Cd content was 14 and 66 μg g−1 for
S1 and S2 soil, respectively, and the Ni and Cr uptake were limited. It has been suggested that hemp has the
ability to avoid cell damage by activating different molecular mechanisms such as the antioxidant system [37].
3.2 Identification and structural features of the GSR gene, a putative heavy metal responsive gene of hemp
Degenerated primers were designed from the coding region after the alignment of GSR related genomes of
different plants. These primer sequences were used to amplify partial cDNA sequences encoding putative GSR
1
gene from hemp leaves. For the identification of the GSR gene, total RNA was extracted from two different
hemp plants and cDNA was synthesized from 2 µg of total RNA from each sample. After PCR, the amplified
PCR product corresponding to the GSR gene from the two different hemp plants’ leaves was analyzed on agarose
gel electrophoresis and a clear band of 215 bp was detected in each sample as shown in lane 1 (hemp plant 1)
and lane 2 (hemp plant 2) in Fig. 1. Amplified PCR products of the GSR gene from two hemp plants’ leaves
were sequenced and a partial sequence of 215 bp was received and identified as a putative GSR gene after the
BLAST search. Characterization of the GSR gene was done with the already identified GSR genes from different
plants. Schematic representation of target GSR included three motifs common to plant GSR representing three
glutathione binding sites, the NADPH binding site and the redox-active disulfite bridge as shown in Fig. 2.
Already published data showed a regulatory role of GSR in influencing the expression of many genes important
in plants' responses to both biotic and abiotic stresses (Table 2). According to a recent study, heavy metals like
Zn, Cu, and Cd essentially increased the activity of GSR involved in the ascorbate-glutathione cycle in sunflower
[38]. In another study, GSR activity increased by 111 and 200% after seven and 14 days, respectively, in roots of
wheat treated with Cd [39]. GSR also showed higher activity under Pb and NaCl toxicity [40]. Luo and his
colleagues also reported the up-regulation of the GSR gene in perennial ryegrass under Cd stress [41]. Thus, up-
regulation of GSR occurs as a defense mechanism against Cd, Cu, Zn and Pb stresses. Therefore, in this study
amplified GSR product from 50 ng cDNA showed higher expression that could be due to heavy metals
accumulation in hemp leaves (Fig. 2).
3.3 Identification and characterization of the PLDα gene, a putative heavy metals responsive gene of hemp
The PLDα gene was amplified by using degenerated primers designed manually from PLDα genome of different
plants aligned by using the multiple alignment program.The amplified PCR product from two different hemp
plants cDNA corresponding to PLDα gene was analyzed on agarose gel electrophoresis and a clear band of 517
bp was detected as shown in lane 1 (hemp plant 1) and lane 2 (hemp plant 2) in Fig. 3. The amplified PLDα gene
was sequenced and then the sequence was analyzed by using bioinformatics tools. The characterization of the
PLDα gene was done by multiple alignments of already published PLDα gene sequences (Fig. 4). Bioinformatics
analysis showed that the Ca2+/phospholipids binding domain, motifs 1 and 2 (catalytic domain) and conserved
evolution boxes A, B and C were found identical to all other plant sequences reported previously. The PLDα
gene was amplified from 50 ng cDNA and a higher expression of the PLDα gene could be observed as shown in
Fig. 3. Higher expression of PLDα gene in this study could be due to heavy metals stress. Previous studies
suggested that low concentrations of heavy metals stimulate PLDα signaling pathways which in turn lead to the
production of ROS hence subsequent cell death accelerated by caspase-like proteases [42]. The PLDα gene has
shown to be involved in ABA responses [43] which play an important role in numerous plant stress responses
like cold, drought and salinity. In another study, a wheat putative PLD-encoding gene showed enhanced gene
expression and thus contributed to the increase in PLD activity under copper stress [44]. Dai and his co-workers
observed PLDb1 mRNA in maize crop in response to Cd stress [45]. In addition, PLDα gene activity increased
in different plants under abiotic stresses (Table 3) [27--29, 46]. This clearly showed the role of PLDα genes in
abiotic stresses. However, to confirm PLDα genes’ role in stress conditions, further studies, particularly
functional studies will be required.
4 Concluding Remarks
1
The present research was conducted to evaluate the phytoremediation potential of the hemp plant and to identify
the genes involved in heavy metals stress tolerance. The results as well as already published data demonstrated
that hemp has a great potential to remove heavy metals, particularly Cu, Cd and Ni from contaminated soils and
could be used in phytoremediation technology. PLDα and GSR are major antioxidant enzymes that protect plant
cells against oxidative damage caused by ROS produced under stress conditions. The identification of PLDα and
GSR genes may provide us with the basic understanding of mechanisms of heavy metals accumulation and
tolerance in plants. The high expression of PLDα and GSR genes in hemp plant leaves indicates that these two
genes express under heavy metals stress in order to help the plant cope with stressful conditions. The
experimental approach used here can prove as a useful tool to characterize PLDα and GSR genes and their
regulation at the transcriptional level. However, more experiments need to be conducted in the future to validate
the results of transcriptional and post-transcriptional changes of PLDα and GSR genes under different heavy
metals concentrations. Moreover, the studies regarding biological activities of GSR and PLDα genes will also be
required to make the detailed elucidation of the physiological functions of these enzymes that play role in heavy
metals stress tolerance. Further studies may lead to the production of heavy metals tolerant varieties of crop
plants through genetic manipulation of the PLDα and GSR genes.
Acknowledgements
This study was financially supported by Higher Education Commission (HEC) of Pakistan through the Startup
Research Grant project.
The authors have declared no conflict of interest.
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Table 1. Accumulation of heavy metals in different parts of Hemp plant in mg/kg
Heavy metal Leaves Roots Shoots Seeds Reference
3.94
2.96
1.03
1.19
[10]
1.3--4.0 [47]
>1000 [48]
50-100 800 50-100 [35]
18.1 ± 8.0(S1)
58.8 ± 26.9 ( S2)
109.2 ± 65.1 (S1)
1368.2 ± 1106.8
(S2)
14 (S1)
66 (S2)
[37]
Cd
151 ± 19 This study
23.20
21.65
1.98
1.69
[10] Pb
39 ± 5 This study
10--12 [49]
1148.16 ± 159.73 0.93 ± 0.08 44.41 ± 3.38 [33]
Cu
1530 ± 19 This study
42--57 [49]
42--94
Zn
4.5 ± 1.2 This study
1.6--6.1 [49]
7.1 ± 1.4 (S1)
31.4 ± 8.1 ( S2)
35.8 ± 18.9(S1 )
321.8 ± 241.6 (S2)
[37]
63.83
63.46
33.24
4.79
[10]
Ni
123 ± 13.2 This study
598--877 [49]
1.4 ± 0.8 (S1)
1.2 ± 0.8 (S2 )
6.2 ± 4.2 (S1)
9.0 ± 9.9 (S2)
[37]
Cr
25.3 ± 1.75 This study
1
Table 2. GSR gene or protein in response to heavy metals in different plants
Heavy metal Plant species Technique Measurement Reference
Zn, Cr, Cu Helianthus annuus RT-PCR, spectrophotometry GSR gene expression and
enzyme activity
[50]
Cd Triticum aestivum RT-PCR, Western-blot
analysis
GSR gene expression and
Protein quantification
[51]
Cd Lolium perenne RT-qPCR GSR gene expression [41]
Cd Populus tremula Glutathione reductase (GR)
assay
GSR enzyme activity [52]
Zn Spinacia oleracea Affinity chromatography GSRenzyme activity [53]
Cd, Cr Nicotiana langsdorffii RT-PCR GSR gene expression [54]
Cd Brassica juncea Glutathione reductase (GR)
assay
GSR enzyme activity [55]
Cu, Cd Arabidopsis thaliana Nothern blotting GSR genes expression [56]
Cd, Cu Helianthus annuus Glutathione reductase assay GSR enzyme activity [57]
Pb Lathyrus sativus Real time RT-PCR GSR gene expression [58]
Table 3. PLDα gene in response to abiotic stresses in different crop plants
Stress Plant species Technique Measurement Reference
Cu Triticum durum RT-PCR, Western
blotting
PLD gene expression and
enzyme activity
[44]
Cd Brassica juncea Northern blot analysis PLD Gene expression [59]
Pb Lathyrus sativus Real time RT-PCR PLD Gene expression [58]
Cd Zea mays Northern blotting PLD Gene expression [45]
Freezing Arabidopsis thaliana Northern blotting PLD Gene expression [60]
Drought Arabidopsis thaliana Immunoblotting PLD protein [61]
Salinity Solanum lycopersicon Western blotting PLD Protein [46]
Drought Zea mays PLD activity assay PLD protein [62]
Cold stress Chorispora bungeana RT-PCR PLD gene expression [27]
Drought Arabidopsis thaliana RT–PCR PLD gene expression [63]
Drought Arabidopsis thaliana Immunoblotting PLD protein [29]
Salt Glycine max qRT-PCR PLD gene expression [28]
1
Figure 1. RT-PCR amplified GSR gene from two different hemp plants. DNA fragments of GSR gene were
amplified from cDNA of two hemp plants and separated using 1.5% agarose gel electrophoresis and visualized
under UV light after staining with ethidium bromide. M: 100 bp DNA ladder marker (Enzynomics, Seoul,
Korea), lane 1 (hemp plant 1) and lane 2 (hemp plant 2): 215 bp GSR gene in two different hemp leaves.
Figure 2. Schematic representation of target GSR gene products. Regions spanning between the vertical arrows
correspond to the deduced proteins from hemp cDNA sequences isolated in the current study.
Figure 3. RT-PCR amplified PLDα gene from hemp leaves. DNA fragments of PLDα gene were separated using
1.5% agarose gel electrophoresis and visualized under UV light after staining with ethidium bromide. M: 100 bp
DNA ladder marker (Enzynomics, Seoul, Korea), lane 1 (hemp plant 1) and lane 2 (hemp plant 2): 517 bp
amplified PLDα gene from two different hemp leaves.
Figure 4. Schematic representation of target PLDα gene products. Regions spanning between the vertical arrows
correspond to the deduced proteins from hemp cDNA sequences isolated in the current study.
1
Figure1
1
Figure2
1
Figure3
1
Figure4
