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Phytoremediation Potential of Hemp (Cannabis sativa L.): Identification and Characterization of Heavy Metals Responsive Genes


Abstract and Figures

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 (1530mgkg-1), Cd (151mgkg-1), and Ni (123mgkg-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 (215bp) and PLDα (517bp) 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.
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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,
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
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:
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: 6 March 2015; Revised: 6 March 2015; Accepted: 15 June 2015
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
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--
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 dierent
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,
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):
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
2.5 Sequence analysis
The PCR product obtained was sent to Laragen sequencing and genotyping (Virginia, CA). Nucleotide sequence
identity was determined using the online BLASTn program ( 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
g1 of Cd, Ni and Cr, respectively. The results revealed that the mean shoot Cd content was 14 and 66 μg g1 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
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
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.
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
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
14 (S1)
66 (S2)
151 ± 19 This study
[10] Pb
39 ± 5 This study
10--12 [49]
1148.16 ± 159.73 0.93 ± 0.08 44.41 ± 3.38 [33]
1530 ± 19 This study
42--57 [49]
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)
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)
25.3 ± 1.75 This study
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
Cd Triticum aestivum RT-PCR, Western-blot
GSR gene expression and
Protein quantification
Cd Lolium perenne RT-qPCR GSR gene expression [41]
Cd Populus tremula Glutathione reductase (GR)
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)
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
PLD gene expression and
enzyme activity
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]
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.
... Hemp (Cannabis sativa L.) and sunflower (Helianthus annuus) plants are known for their tolerance to elevated heavy metals in soils and their ability to bioaccumulate these pollutants (Stoikou et al. 2017;Husain et al. 2019). Hence, their use in bioremediation strategies is largely discussed (Ahmad et al. 2016). Therefore, the use of hemp seeds, sunflower kernels but also peanuts and their derived products raise questions about potentially negative health effects. ...
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Nickel is a food contaminant of natural or anthropogenic origin. Monitoring of contaminants in food in general allows obtaining an overview on the presence of substances that are undesirable to health. The aim of this study was to analyse nickel content in food of non-animal origin and beverages sold in Luxembourg to determine the exposure of the population to this contaminant. In total, 660 samples were analysed in the timeframe from 2017 to 2021. The results demonstrate high concentrations of nickel in cashew nuts, walnuts, hemp and sunflower seeds, dried peas, oregano, and cocoa powder. Surveillance of contaminants in food allows identifying contributors to the chronic and acute exposure of nickel in order to potentially set official maximum levels in European legislation in the future, allowing for better enforcement actions in case of contaminated products and increasing consumer protection.
... Cannabis for medicinal purposes has been partially or fully legalized in 36 countries [14]. A hemp renaissance within last three decades has led to extensive research work on different aspects of its development such as agronomy [15], industrial properties [16], energy source [17], environmental benefits [18], suitability assessment [13,19], pharmaceutical value [20,21,22] and many more [23]. ...
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Hemp (Cannabis sativa L.) is a multi-million-dollar industry in several temperate countries. In South and Southeast Asian region, it remains a neglected and underutilised due to several legal, political, and cultural barriers. Therefore, very limited research has been done on value chain of hemp in this region. Nevertheless, as discussions are ongoing on the legalization of hemp in some of the countries in the region, interest in research and development of hemp is growing. The objective of this review is to identify what has been done on hemp in the region and outline the potentials and challenges in adopting hemp as an industrial crop in tropical South and Southeast Asia. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used to select and review research articles. Out of the 12,210 studies, 36 were selected for review and analysis. The results demonstrate the potential of hemp in the South and Southeast Asian region in terms of genetic diversity, growth habits, environmental and health benefits, and value-added products. To motivate the commercial cultivation, several key aspects were identified that includes development of region/location specific cultivars, introduction of site/cultivar specific management practices and development of proper market facilities. The review concludes that hemp can be a potential candidate for crop diversification across South and Southeast Asia.
... the release of secondary metabolites (e.g. nonintoxicating phytocannabinoids, terpenes, and phenolic compounds, see Schluttenhofer & Yuan, 2017 Ahmad et al. 2016) is often reported. We hypothesized that hemp canvas will act as a homogeneous physical barrier restricting weeds access to light and changing to soil properties, but also will act on the soil microbiota through the release of secondary metabolites and carbon during its biodegradation. ...
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Aims - Plastic films are used to mulch soils to control weeds, especially in organic farming. Their application leaves persistent plastic fragments in soils, with poorly understood environmental and health consequences. Plant fiber textiles (bio-canvas) are promising alternatives since they are more persistent than straw mulching and are entirely biodegradable. Hemp fibers are particularly interesting materials due to their renowned resistance, allelopathic and trophic properties for soil life. However, their effects on soil microbiota and yield remain unclear. Methods - In a greenhouse experiment, we assessed the effect of soil mulching (bare soil control, plastic mulch, hemp straw mulching, hemp-canvas) on lettuce growth, soil climatic conditions, enzymatic activities and microbial communities (bacteria and fungi). Our experiment allowed to distinguish effects associated to mulching, being i) the homogeneity of soil covering (plastic mulch and hemp canvas) or not (control, hemp mulch), ii) the biodegradability (hemp mulch, hemp canvas) or not (control, plastic mulch), and iii) their interaction. Results - An interaction occurred between cover homogeneity and biodegradability when using the hemp canvas, leading to higher soil relative water content, stable soil temperature, higher laccase and arylamidase activities, and different soil microbial community structures and fungal diversity, with comparable lettuce yields to that obtained with plastic mulch. Plastic cover induced higher soil temperatures, lower enzymatic activities, and different soil microbial community structures. Conclusions - We conclude that hemp canvas secures lettuce yields, but through different mechanisms compared to plastic mulch, notably via a biostimulating effect on soil microbial diversity and functioning.
... Therefore, Cu can be highly accumulated in the root system architecture compared to the upper parts of the plant. This is further supported by the achievements of other studies that reported a maximum copper accumulation of 0.01, 1.5, and 0.03 mg·g −1 of C. sativa L. in flowers, roots, and shoots, respectively [76][77][78]. ...
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This study proposes the phytoremediation of phenanthrene (PHE)-, pyrene (PYR)-, and copper (Cu)-contaminated soil by Cannabis sativa L. The experimental campaign was conducted in 300 mL volume pots over a 50 d period using different initial polycyclic aromatic hydrocarbon (PAH) concentrations, i.e., 100 (PC1), 200 (PC2), and 300 (PC3) mg ƩPAHs kg−1 dry weight of soil, while maintaining a constant Cu concentration of 350 mg∙kg−1. PHE and PYR removal was 93 and 61%, 98 and 48%, and 97 and 36% in PC1, PC2, and PC3, respectively, in the greenhouse condition. The highest Cu extraction amounted to 58 mg∙kg−1. In general, the growth of C. sativa L. under the PC1, PC2, and PC3 conditions decreased by approximately 25, 65, and 71% (dry biomass), respectively, compared to the uncontaminated control. The present study is aimed at highlighting the phytoremediation potential of C. sativa L. and providing the preliminary results necessary for future field-scale investigations.
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Soil contamination by heavy metals, such as zinc, has significant environmental consequences. Phytoremediation, among various remediation techniques, has been developed and applied to restore contaminated soils. However, phytoremediation has limitations related to slow plant growth, influenced by contaminant toxicity. This study aims to investigate the effect of the interaction between zinc and silicon on growth, hydration, photosynthetic and biochemical behaviours, as well as the phytoremediation capacity in oleander (Nerium oleander), a promising species for phytoremediation. Oleander plants were exposed to four treatments, including two adequate zinc treatments (0.76 µM Zn) with two different silicon concentrations (0 mM and 0.5 mM Si) and two zinc toxic treatments (1800 µM Zn) with the same silicon concentrations (0 mM and 0.5 mM Si). The results revealed that zinc toxicity negatively affected most of the measured parameters. However, the depressive effects of zinc toxicity were significantly mitigated under silicon treatments. Adding silicon alleviated leaf chlorosis and improved biomass production, water status, photosynthetic pigment contents, photosynthetic gas exchange, oxidation state of photosystem I (PSI), membrane integrity, soluble protein content, and antioxidant enzyme activity (especially guaiacol peroxidase). Overall, silicon had beneficial effects on the phytoremediation capacity of N. oleander. Therefore, fertilization rich in silicon could represent an effective solution for enhancing the phytoremediation capacity of this species, minimizing one of the major disadvantages of phytoremediation, namely low biomass production influenced by toxicity.
Cannabis sativa is a widely dispersed plant that may be found in a broad range of habitats, elevations, and climatic conditions all over the globe. It is a member of the Cannabis genus. Historically, it has been used by humans for more than 5000 years, making it one of the oldest plant sources of food and textile fibres. Originating in Western Asia and Egypt, the cultivation of Cannabis sativa for textile fibre later spread to Europe, and in 1606, the cultivation of hemp was brought to North America, where it has remained since. For the majority of the 18th and 19th centuries, hemp had an important economic role in Europe, primarily in the manufacturing of ropes and textiles. Cannabis sativa L. (marijuana; Cannabaceae) is a popular plant with extensive distribution that produces fibre and food, as well as a psychoactive drug. Hemp seed has traditionally been harvested for the oil that can be derived from it and used in a variety of applications, including culinary and the production of soaps, paints, lubricants, and cosmetics. Furthermore, hemp has long been used as a medical plant.
Phytoremediation is a plant-based technology that is both cost-effective and environmentally beneficial, and it is classified as an in-situ modification method. Using phytoremediation, on the other hand, results in the restoration of the preservation site's biological activity, physical structure, and chemical properties. Heavy metals can be found in a variety of places in the environment. Because most of these compounds are water soluble, they circulate quickly across the environment. Heavy metals and other pollutants in high concentrations can impair the body's physiology and biochemistry, resulting in health problems. This chapter will review the literature available in different data bases on Cannabis sativa for possible remediation of pollutants from different ecosystems.
The diverse activities of humans have changed the composition and organization of soil which have resulted in the contamination of our environment. A number of methods have been used to get rid of these contaminants from our environment, but majority of the methods are expensive and non-effective and do not give desired results. The technique of phytoremediation includes the use of either plants or plant products to clean the contaminated sites. Phytoremediation takes advantage of plant’s natural ability to take up, collect, store, or degrade the inorganic and organic substances. It is an economical and natural green technology, which helps us in removing the toxic elements from our environment by making use of wild weeds or small herbal plants. It offers a promising tool for the hyperaccumulation of various heavy metals such as lead, nickel, chromium, arsenic, mercury, etc. Thus, plants having an inherent capacity to accumulate heavy metals in their roots or their shoots can form phytochelates and can convert toxic metals into stable compounds.
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Industrial wastewater irrigation of agriculture crops could cause a lot of environmental and health problems in developing countries due to heavy metals deposition in agriculture soils as well as edible plants consumption by human beings. Therefore, this study was conducted to find out heavy metals’ concentration in industrial wastewater and soil irrigated with that wastewater. In addition, the impact of industrial wastewater irrigation on Parthenium hysterophorus and Zea mays genes involved in growth improvement and inhibition of selected plants. For this purpose, plant samples from agriculture fields irrigated with wastewater from Hattar Industrial Estate (HIE) of Haripur, Pakistan and control plants from non-contaminated soil irrigated with tape water were collected after fifteen and forty-five days of germination. Heavy metals concentration in the collected plant samples, wastewater and soil were determined and results revealed that the study area was predominantly contaminated with Cr, Pb, Ni, Cu, Co, Zn and Cd concentrations of 38.98, 21.14, 46.01, 155.73, 12.50, 68.50 and Cd 7.01 mg/kg, respectively. The concentrations of these heavy metal have surpassed permissible limit of these metals in normal agriculture soil. Expansins and cystatin (plant growth enhancers), metacaspases (plant growth inhibitor) genes expression were studied through reverse transcription polymerase chain reaction and results showed that the expression of these genes was higher in samples collected from wastewater-irrigated soils as compared to control. More expression of these genes was observed in 45 days old samples as compared to 15 days old samples and control. Taken together, this study suggests the use of Parthenium and maize for phytoremediation, however, they should not be used for eating purposes if irrigated with industrial wastewater.
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This study's aim is to be a continuation of the research conducted by Mihoc et al, 2012 to deternine the heavy metal content for the hemp seeds of Five romanian varieties by following the distribution of Fe, Mn, In, Cu and Ni in the two fractions: hulled hemp seeds and shell as opposed to whole hemp seeds. Microelements content in hemp seeds are: Fe (130-164 mg/kg), Mn (89-108 mg/kg), In (42-57 mg/kg), Cu (10-12 mg/kg), Ni (1.6-6.1 mg/kg), Cr (598-877μ/kg) and Mo (265-652μg/kg). The Pb analysis shows a high dispersion of the results with many values under the quantification limit. Therefore the Pb concentration in hemp seeeds belongs to the range of 217-626 fig/kg excepting the Armanca variety. Iron is concentrated in shell, zinc and nickel in hemp heart, while manganese and copper are equally balanced in both the core and the shell.
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Phytoremediation is a site remediation strategy, which employs plants to remove non-volatile and immisible soil contents. This sustainable and inexpensive process is emerging as a viable alternative to traditional contaminated land remediation methods. To enhance phytoremediation as a viable strategy, fast growing plants with high metal uptake ability and rapid biomass gain are needed. This paper provides a brief review of studies in the area of phytoaccumulation, most of which have been carried out in Europe and the USA. Particular attention is given to the role of phytochelators in making the heavy metals bio-available to the plant and their symbionts in enhancing the uptake of bio-available heavy metals.
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Leachate derived from bioleaching process contains high amount of metals that must be removed before discharging the water. Aspergillus fumigatus was isolated from a gold mine tailings and its ability to remove of As, Fe, Mn, Pb, and Zn from aqueous solutions and leachate of bioleaching processes was assessed. Batch sorption experiments were carried out to characterize the capability of fungal biomass (FB) and iron coated fungal biomass (ICFB) to remove metal ions in single and multi-solute systems. The maximum sorption capacity of FB for As(III), As(V), Fe, Mn, Pb, and Zn were 11.2, 8.57, 94.33, 53.47, 43.66, and 70.4 mg/g, respectively, at pH 6. For ICFB, these values were 88.5, 81.3, 98.03, 66.2, 50.25, and 74.07 mg/g. Results showed that only ICFB was found to be more effective in removing metal ions from the leachate. The amount of adsorbed metals from the leachate was 2.88, 21.20, 1.91, 0.1, and 0.08 mg/g for As, Fe, Mn, Zn, and Pb, respectively. The FT-IR analysis showed involvement of the functional groups of the FB in the metal ions sorption. Scanning electron microscopy revealed that surface morphological changed following metal ions adsorption. The study showed that the indigenous fungus A. fumigatus was able to remove As, Fe, Mn, Pb, and Zn from the leachate of gold mine tailings and therefore the potential for removing metal ions from metal-bearing leachate.
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An in vitro experiment was conducted to elucidate the role of polyamines and phospholipase D (PLD) in maize (Zea mays L.) response to drought stress simulated by PEG-6000. During the early stage of drought stress, an increase of PLD activity played a major role in stomatal closure as an adaptive response to drought stress and this process was independent of polyamine synthesis. Under prolonged drought stress, the total content of polyamines including putrescine (Put), spermidine (Spd) and spermine (Spm) increased at 120 min after drought stress treatment while the ratio of Spd + Spm/Put decreased, which caused an increase in PLD activity, relative membrane permeability and malondialdehyde content (MDA). Exogenous polyamines enhanced PLD activity, and the greatest effect was induced by Put. Therefore, we concluded that PLD played an important role in mitigation of drought stress damage in the early stage of drought stress which was independent of polyamine accumulation. However, in the later stage of drought stress, the obvious elevation of PLD activity led to serious membrane damage regulated by the ratio of Spd + Spm/Put.
Phospholipase D (PLD), which hydrolyzes phospholipids into free head groups and phosphatidic acid (PA), may regulate cellular processes through the production of lipid and lipid-derived messengers. We have genetically abrogated PLDα, the most prevalent isoform of PLD in plants, and the depletion of PLDα in Arabidopsis decreased the levels of PA and superoxide production in Arabidopsis leaf extracts. Addition of PA promoted the synthesis of superoxide in the PLDα-depleted plants, as measured by chemiluminescence and superoxide dismutase-inhibitable, NADPH-dependent reduction of cytochrome c and nitroblue tetrazolium. The PA-enhanced generation of superoxide was associated mainly with microsomal membranes. Among various lipids tested, PA was the most effective stimulator with the optimal concentrations between 100 and 200 μm. The PA-promoted production of superoxide was observed also in leaves directly infiltrated with PA. The added PA was more effective in stimulating superoxide generation in the PLDα-depleted leaves than in the PLDα-containing, wild-type leaves, suggesting that PA produced in the cell was more effective than added PA in promoting superoxide production. These data indicate that PLD plays a role in mediating superoxide production in plants through the generation of PA as a lipid messenger.
Multiple forms of phospholipase D (PLD) were activated in response to wounding, and the expressions of PLDα, PLDβ, and PLDγ differed in wounded Arabidopsis leaves. Antisense abrogation of the common plant PLD, PLDα, decreased the wound induction of phosphatidic acid, jasmonic acid (JA), and a JA-regulated gene for vegetative storage protein. Examination of the genes involved in the initial steps of oxylipin synthesis revealed that abrogation of the PLDα attenuated the wound-induced expression of lipoxygenase 2 (LOX2) but had no effect on allene oxide synthase (AOS) or hydroperoxide lyase in wounded leaves. The systemic induction of LOX2, AOS, and vegetative storage protein was lower in the PLDα-suppressed plants than in wild-type plants, with AOS exhibiting a distinct pattern. These results indicate that activation of PLD mediates wound induction of JA and that LOX2 is probably a downstream target through which PLD promotes the production of JA.
The effects of double stress environment i.e. lead (heavy metal) and NaCl (saline) on the activity of antioxidant enzymes in Vigna radiata seedling were studied. The antioxidant activities of enzymes, i.e of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and their activity proportions were examined. Superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase and glutathione reductase activities were substantially increased in a combined stress environment as compared to catalase. Further, in comparison with catalase and ascorbate peroxidase, glutathione reductase showed increased activities together with superoxide dismutase in a combined stress environment. Superoxide dismutase and glutathione reductase showed higher activity proportion in combined treatment. Physiological role of these enzymes in stress tolerance mechanism is discussed.
Phytoremediation is the use of plants to extract, sequester or mineralize pollutants. This process is seen as an ecologically sound strategy for management of contaminated ecosystems. In this review, current status of several subsets of phytoremediation are discussed which includes: (a) Phytoextraction – which is a process in which high biomass pollutant accumulating plants are used to accumulate and transport pollutants from the soil to harvestable parts of plants. (b) Phytofiltration – which is a process in which plant roots are used to precipitate and concentrate pollutants from effluents. (c) Phytostabilization -here plants stabilize pollutants, thus rendering them harmless. (d) Phytovolatilization –plants absorb pollutants and convert them into gaseous components via transpiration. The advantages inherent in these technologies are also discussed. There is need for further understanding on the processes that affect pollutant availability, rhizosphere processes, pollutant uptake and sequestration.
We conducted an analysis of heavy metals content, including As, Cd, Cr, Cu, Hg, Pb, and Zn in sediments from aquatic ecosystems in China measured in recent publications. Then, we evaluated the extent of heavy metal pollution in these ecosystems in seven different industrial districts in China (Dongbei, Huabei, Huazhong, Huanan, Huaxik, Xibei, and Huadong) with the potential ecological risk index. We found that Cd was the most concentrated pollutant, followed by Hg and As, while Cr, Cu, Pb, Zn were found in low concentrations in sediments from all types of aquatic ecosystem in China. Sediments collected from all seven industrial districts were heavily polluted, and the sequence, from most to least polluted was Dongbei>Huabei>Huazhong>Huanan>Huaxi>Xibei>Huadong. All four types of aquatic ecosystem were found to be seriously polluted and the sequence, from most to least polluted was: river>sea>lake>wetland. Specifically, Cd and Hg were the most serious pollutants in all four aquatic ecosystems, and As was also a serious pollutant in rivers. For the seven industrial districts studied the sea was the most polluted ecosystem in Dongbei, the river was the most polluted ecosystem in Huabei, Huanan, Huazhong, and the lake was the most polluted in Huadong, Huaxi, and Xibei.
The influence of freezing on phospholipase D (PLD) was studied in Chorispora bungeana Fisch. & C.A. Mey., which is a naturally cold-tolerant species. During the freezing treatment (−4 °C), PLD activities in both microsomal and mitochondrial membranes increased at day 3, remained at a high level at day 6 and then declined to a moderate level. The RT-PCR analyses showed that PLD activity partially corresponded to the CbPLD gene transcript level. The freezing treatment resulted in increases in the K m and V max for microsomal and mitochondrial PLD, respectively. Freezing injury, as measured by electrolyte leakage and malondialdehyde content, peaked at day 6 and then gradually decreased. Alleviation of freezing injury was related to a decreased content of membrane-associated Ca2+. We suggest that the specific mechanism of cold resistance of C. bungeana is linked with PLD.