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

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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,
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
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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
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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 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,
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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
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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
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
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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
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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.
References
[1] F. Arik, T. Yaldiz, Heavy Metal Determination and Pollution of the Soil and Plants of Southeast Tavsanli
(Kutahya, Turkey), Clean -- Soil Air Water 2010, 38, 1017--1030.
[2] B. J. Seh-Bardan, R. Othman, S. Abd Wahid, F. Sadegh-Zadeh, A. Husin, Biosorption of Heavy Metals in
Leachate Derived from Gold Mine Tailings Using Aspergillus fumigatus, Clean -- Soil Air Water
2013,41, 356--364.
[3] L. Zhang, H. Shao, Heavy Metal Pollution in Sediments from Aquatic Ecosystems in China, Clean -- Soil Air
Water 2013, 41, 878--882.
[4] R. B. Meagher, Phytoremediation of toxic elemental and organic pollutants, Curr. Opin. Plant Biol. 2000, 3,
153--162.
[5] C. O. Nwoko, Trends in phytoremediation of toxic elemental and organic pollutants, Afr. J. Biotechnol.
2010, 9, 6010--6016.
[6] A. G. Khan, C. Kuek, T. M. Chaudhry, C. S. Khoo, W. J. Hayes, Role of plants, mycorrhizae and
phytochelators in heavy metal contaminated land remediation, Chemosphere 2000, 41, 197--207.
[7] J. L. Freeman, M. W. Persans, K. Nieman, C. Albrecht, W. Peer, I. J. Pickering, D. E. Salt, Increased
glutathione biosynthesis plays a role in nickel tolerance in Thlaspi nickel hyperaccumulators, Plant Cell
2004, 16, 2176--2191.
[8] S. Aubert, J. P. Schwitzguébel, Screening of plant species for the phytotreatment of wastewater containing
sulphonated anthraquinones, Water Res. 2004, 38, 3569--3575.
1
[9] S. Citterio, N. Prato, P. Fumagalli, R. Aina, N. Massa, A. Santagostino, S. Sgorbati, et al., The arbuscular
mycorrhizal fungus Glomus mosseae induces growth and metal accumulation changes in Cannabis
sativa L., Chemosphere 2005, 59, 21--29.
[10] P. Linger, J. Mussig, H. Fischer, J. Kobert, Industrial hemp (Cannabis sativa L.) growing on heavy metal
contaminated soil: fibre quality and phytoremediation potential, Ind. Crop. Prod. 2002, 16, 33--42.
[11] B. Kos, D. Leštan, Induced phytoextraction/soil washing of lead using biodegradable chelate and permeable
barriers, Environ. Sci. Technol. 2003, 37, 624--629.
[12] K. J. A. Davies, Intracellular proteolytic systems may function as secondary antioxidant defenses: An
hypothesis, J. Free Radicals Biol. Med. 1986, 2, 155--173.
[13] R. Ahmad, Y. Zuily-Fodil, C. Passaquet, O. Bethenod, R. Roche, A. Repellin, Ozone and aging up-regulate
type II metacaspase gene expression and global metacaspase activity in the leaves of field-grown maize
(Zea mays L.) plants, Chemosphere 2012, 87, 789--795.
[14] L. Gomez, G. Noctor, M. Knight, C. Foyer, Regulation of calcium signaling and gene expression by
glutathione, J. Exp. Bot. 2004, 55, 1851--1859.
[15] C. H. Foyer, G. Noctor, Redox homeostasis and antioxidant signaling: a metabolic interface between stress
perception and physiological responses, Plant Cell Online 2005, 17, 1866--1875.
[16] G. G. Yannarelli, A. J. Fernández-Alvarez, D. M. Santa-Cruz, M. L. Tomaro, Glutathione reductase activity
and isoforms in leaves and roots of wheat plants subjected to cadmium stress, Phytochemistry2007, 68,
505--512.
[17] M. Liscovitch, M. Czarny, G. Fiucci, X. Q. Tang, Phospholipase D: molecular and cell biology of a novel
gene family, Biochem. J. 2000, 345, 401--415.
[18] N. Divecha, R. F. Irvine, Phospholipid signaling, Cell 1995, 80, 269--278.
[19] D. English, Y. Cui, R. A. Siddiqui, Messenger functions of phosphatidic acid, Chemistry and physics of
lipids 1996, 80, 117--132.
[20] A. M. Laxalt, B. ter Riet, J. C. Verdonk, L. Parigi, W. I. Tameling, J. Vossen, M. Haring, et al.,
Characterization of five tomato phospholipase D cDNAs: rapid and specific expression of LePLDβ1 on
elicitation with xylanase, Plant J. 2001, 26, 237--247.
[21] T. Munnik, J. A. Van Himbergen, B. ter Riet, F. J. Braun, R. F. Irvine, H. van den Ende, A. Musgrave,
Detailed analysis of the turnover of polyphosphoinositides and phosphatidic acid upon activation of
phospholipases C and D in Chlamydomonas cells treated with non-permeabilizing concentrations of
mastoparan, Planta 1998, 207, 133--145.
[22] K. Pappan, X. Wang, Plant Phospholipase Dα Is an Acidic Phospholipase Active at Near-Physiological Ca2+
concentrations, Arch. Biochem. Biophys.1999, 368, 347--353.
[23] M. Li, R. Welti, X. Wang, Quantitative profiling of Arabidopsis polar glycerolipids in response to
phosphorus starvation. Roles of phospholipases Dζ1 and Dζ2 in phosphatidylcholine hydrolysis and
digalactosyldiacylglycerol accumulation in phosphorus-starved plants, Plant Physiol. 2006, 142, 750--
761.
[24] Y. Hong, S. P. Devaiah, S. C. Bahn, B. N. Thamasandra, M. Li, R. Welti, X. Wang, Phospholipase Dε and
phosphatidic acid enhance Arabidopsis nitrogen signaling and growth, Plant J. 2009, 58, 376-387.
[25] X. Wang, Plant phospholipases, Ann. Rev. Plant Biol. 2001, 52, 211--231.
[26] C. Wang, C. A. Zien, M. Afitlhile, R. Welti, D. F. Hildebrand, X. Wang, Involvement of phospholipase D in
1
wound-induced accumulation of jasmonic acid in Arabidopsis, Plant Cell Online 2000, 12, 2237--2246.
[27] N. Yang, X. L. Yue, H. Zhang, G. F. Wu, F. X. Ding, T. G. Zhang, L. Z. An, Characterization of
phospholipase D from Chorispora bungeana callus in response to freezing stress, Biol. Plant.2013, 57,
113--120.
[28] J. Zhao, D. Zhou, Q. Zhang, W. Zhang, Genomic analysis of phospholipase D family and characterization
of GmPLDαs in soybean (Glycine max), J. Plant Res. 2012, 125, 569--578.
[29] Y. Sang, S. Zheng, W. Li, B. Huang, X. Wang, Regulation of plant water loss by manipulating the
expression of phospholipase Dα, Plant J. 2001, 28, 135--144.
[30] B. O. Bargmann, A. M. Laxalt, B. ter Riet, B. van Schooten, E. Merquiol, C. Testerink, M. A. Haring, et al.,
Multiple PLDs required for high salinity and water deficit tolerance in plants, Plant Cell Physiol. 2009,
50, 78--89.
[31] A. Baker, R. Brooks, Terrestrial higher plants which hyperaccumulate metallic elements. A review of their
distribution, ecology and phytochemistry, Biorecovery1989, 1, 81--126.
[32] H. Dahmani-Muller, F. van Oort, B. Gélie, M. Balabane, Strategies of heavy metal uptake by three plant
species growing near a metal smelter, Environ. Pollut.2000, 109, 231--238.
[33] E. Bona, F. Marsano, M. Cavaletto, G. Berta, Proteomic characterization of copper stress response in
Cannabis sativa roots, Proteomics 2007, 7, 1121--1130.
[34] G. R. Shi, Q. S. Cai, Cadmium tolerance and accumulation in eight potential energy crops, Biotechnol. Adv.
2009, 27, 555--561.
[35] P. Linger, A. Ostwald, J. Haensler, Cannabis sativa L. growing on heavy metal contaminated soil: growth,
cadmium uptake and photosynthesis, Biol. Plant. 2005, 49, 567--576.
[36] G. R. Shi, Q. S. Cai, Zinc tolerance and accumulation in eight oil crops, J. Plant Nutr. 2010, 33, 982-997.
[37] S. Citterio, A. Santagostino, P. Fumagalli, N. Prato, P. Ranalli, S. Sgorbati, Heavy metal tolerance and
accumulation of Cd, Cr and Ni by Cannabis sativa L., Plant Soil 2003, 256, 243--252.
[38] E. Nehnevajova, L. Lyubenova, R. Herzig, P. Schröder, J. P. Schwitzguébel, T. Schmülling, Metal
accumulation and response of antioxidant enzymes in seedlings and adult sunflower mutants with
improved metal removal traits on a metal-contaminated soil, Environ. Exp. Bot. 2012, 76, 39--48.
[39] G. G. Yannarelli, A. J. Fernández-Alvarez, D. M. Santa-Cruz, M. L. Tomaro, Glutathione reductase activity
and isoforms in leaves and roots of wheat plants subjected to cadmium stress, Phytochemistry2007, 68,
505-512.
[40] Z. S. Siddiqui, Effects of double stress on antioxidant enzyme activity in Vigna radiata (L.) Wilczek, Acta
Bot. Croatica 2013, 72, 145–156.
[41] H. Luo, H. Li, X. Zhang, J. Fu, Antioxidant responses and gene expression in perennial ryegrass (Lolium
perenne L.) under cadmium stress, Ecotoxicology 2011, 20, 770--778.
[42] E. T. Yakimova, V. M. Kapchina-Toteva, E. J. Woltering, Signal transduction events in aluminum-induced
cell death in tomato suspension cells, J. Plant Physiol. 2007, 164, 702--708.
[43] S. Ritchie, S. Gilroy, Abscisic acid signal transduction in the barley aleurone is mediated by phospholipase
D activity, Proc. Natl. Acad. Sci. 1998, 95, 2697--2702.
[44] F. Navari-Izzo, B. Cestone, A. Cavallini, L. Natali, T. Giordani, M. F. Quartacci, Copper excess triggers
phospholipase D activity in wheat roots, Phytochemistry 2006, 67, 1232--1242.
[45] Q. L. Dai, B. F. Huang, Z. Y. Yang, J. G. Yuan, J. Z. Yang, Identification of cadmium-induced genes in
1
maize seedlings by suppression subtractive hybridization, Front. Environ. Sci. Eng. China 2010, 4, 449-
-458.
[46] B. O. Bargmann, A. M. Laxalt, B. ter Riet, B. van Schooten, E. Merquiol, C. Testerink, M. A. Haring, et al.,
Multiple PLDs required for high salinity and water deficit tolerance in plants, Plant Cell Physiol. 2009,
50, 78--89.
[47] M. Mihoc, G. Pop, E. Alexa, I. Radulov, Nutritive quality of romanian hemp varieties (Cannabis sativa L.)
with special focus on oil and metal contents of seeds, Chem. Cent. J. 2012, 6, 1--12.
[48] G. Shi, Q. Cai, Cadmium tolerance and accumulation in eight potential energy crops, Biotechnol. Adv.
2009, 27, 555--561.
[49] M. Mihoc, G. Pop, E. Alexa, D. Dem, A. Militaru, Microelements distribution in whole Hemp seeds
(Cannabis sativa L.) and in their fractions, Rev. Chim. 2013, 64, 776--780.
[50] E. Nehnevajova, L. Lyubenova, R. Herzig, P. Schröder, J. P. Schwitzguébel, T. Schmülling, Metal
accumulation and response of antioxidant enzymes in seedlings and adult sunflower mutants with
improved metal removal traits on a metal-contaminated soil, Environ. Exp. Bot. 2012, 76, 39--48.
[51] G. G. Yannarelli, A. J. Fernández-Alvarez, D. M. Santa-Cruz, M. L. Tomaro, Glutathione reductase activity
and isoforms in leaves and roots of wheat plants subjected to cadmium stress, Phytochemistry2007, 68,
505--512.
[52] P. Kieffer, P. Schröder, J. Dommes, L. Hoffmann, J. Renaut, J. F. Hausman, Proteomic and enzymatic
response of poplar to cadmium stress, J. Proteomics 2009, 72, 379--396.
[53] B. Halliwell, C. Foyer, Properties and physiological function of a glutathione reductase purified from
spinach leaves by affinity chromatography, Planta 1978, 139, 9--17.
[54] R. Fuoco, P. Bogani, G. Capodaglio, M. Del Bubba, O. Abollino, S. Giannarelli, M. M. Spiriti, et al.,
Response to metal stress of Nicotiana langsdorffiiplants wild-type and transgenic for the rat
glucocorticoid receptor gene, J. Plant Physiol. 2013, 170, 668--675.
[55] Y. Markovska, N. Gorinova, M. Nedkovska, K. Miteva, Cadmium-induced oxidative damage and
antioxidant responses in Brassica juncea plants, Biol. Plant. 2009, 53, 151--154.
[56] C. Xiang, D. J. Oliver, Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid
in Arabidopsis, Plant Cell Online 1998, 10, 1539--1550.
[57] S. M. Gallego, M. P. Benavídes, M. L. Tomaro, Effect of heavy metal ion excess on sunflower leaves:
evidence for involvement of oxidative stress, Plant Sci. 1996, 121, 151--159.
[58] J. Brunet, G. Varrault, Y. Zuily-Fodil, A. Repellin, Accumulation of lead in the roots of grass pea (Lathyrus
sativusL.) plants triggers systemic variation in gene expression in the shoots, Chemosphere 2009, 77,
1113--1120.
[59] L. Minglin, Y. X. Zhang, T. Y. Chai, Identification of genes up-regulated in response to Cd exposure, in
Brassica juncea L., Gene 2005, 363, 151--158.
[60] R. Welti, W. Q. Li, M. Y. Li, Y. M. Sang, H. Biesiada, H. E. Zhou, C. B. Rajashekar, et al., Profiling
membrane lipids in plant stress responses-Role of phospholipase D alpha in freezing-induced lipid
changes in Arabidopsis, J. Biol. Chem. 2002, 277, 31994--32002.
[61] Y. Sang, D. Cui, X. Wang, Phospholipase D and phosphatidic acid-mediated generation of superoxide in
Arabidopsis, Plant Physiol. 2001, 126, 1449--1458.
[62] Z. F. An, C. Y. Li, L. X. Zhang, A. K. Alva, Role of polyamines and phospholipase D in maize (Zea mays
1
L.) response to drought stress, South Afr. J. Bot. 2012, 83, 145--150.
[63] S. P. Mane, C. Vasquez-Robinet, A. A. Sioson, L. S. Heath, R. Grene, Early PLDα-mediated events in
response to progressive drought stress in Arabidopsis: a transcriptome analysis, J. Exp. Bot. 2007, 58,
241--252.
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
... It can adapt to different soil conditions and grows in a variety of climates [36]. Many studies showed that hemp has a high tolerance to metals [52]. Industrial hemp can often take up metals and store them in different parts of the plant, with no detriment to the plant itself (Table 1) [36,53,54]. ...
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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.
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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.
Article
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.
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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.
Article
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.
Article
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.