Ecotoxicology and Environmental Safety 171 (2019) 54–65

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Over-expression of CarMT gene modulates the physiological performance T

and antioxidant defense system to provide tolerance against drought stress
in Arabidopsis thaliana L
Arvind Kumar Dubeya,b, Navin Kumara, Anil Kumara,b, Mohd Akram Ansaria,b, Ruma Ranjana,
Ambedkar Gautama, Meenakshia,b, Nayan Sahuc, Vivek Pandeya, Soumit Kumar Beheraa,
⁎
Shekhar Mallicka, Veena Pandeb, Indraneel Sanyala,
a
CSIR-National Botanical Research Institute, Lucknow, India
b
Department of Biotechnology, Kumaun University, Bhimtal Campus, Nainital, India
c
Department of Botany, Indira Gandhi National Tribal University, Amarkantak, Madhya Pradesh, India

A R T I C LE I N FO A B S T R A C T

Keywords: Drought is one of the major abiotic stresses which negatively affect plant growth and crop yield. Metallothionein
Arabidopsis (MTs) is a low molecular weight protein, mainly involved in metal homeostasis, while, its role in drought stress is
Chickpea still to be largely explored. The present study was aimed to investigate the role of MT gene against drought stress.
Drought The chickpea MT based on its up-regulation under drought stress was overexpressed in Arabidopsis thaliana to
Metallothionein
explore its role in mitigation of drought stress. The total transcript of MT gene was up to 30 fold higher in
Physiological performance
ROS
transgenic lines. Arabidopsis plants transformed with MT gene showed longer roots, better efficiency of survival
and germination, larger siliques and higher biomass compared to WT. The physiological variables (A, WUE, G, E,
qP and ETR) of WT plants were reduced during drought stress which recovered in transgenic Arabidopsis lines.
The enzymatic and non-enzymatic antioxidant (APX, GPX, POD, GR, GRX, GST, CAT, MDHAR, ASc and GSH)
levels were also enhanced in transgenic lines to provide tolerance. Simultaneously, drought responsive amino
acids, i.e. proline and cysteine contents were higher in transgenic lines. Overall, the results suggest that MT gene
is actively involved in the mitigation of drought stress and could be the choice for genetic engineering strategy to
overcome drought stress.

1. Introduction low-temperature influences the expression of MTs in plants (Dunaeva

Metallothioneins (MTs) are metal-binding proteins; conserved in all
the living organisms, first isolated 40 years ago from horse kidney
(Cobbett and Goldsbrough, 2002). The exact function of MTs is yet to
be explored. However, considering the different evidences, it is known
to play a crucial role in metal homeostasis and detoxification processes.
The four isoforms of MTs are classified on the basis of metal binding
cysteine (Cys) residues, containing Cys-Cys, Cys-Xaa-Cys or Cys-Xaa-
Xaa-Cys, which furnished sulphydryl ligands for coordination of diva-
lent metal ions. In plants, MTs are organ-specific, in which transcript of
MT1 has been identified in roots. However, the MT2 transcript has been
reported in leaves, while MT3 and MT4, in ripening fruit and germi-
nating seed, respectively (Dabrowska, 2012). Under different stress
conditions, MTs function as efficient scavengers of reactive oxygen
species (ROS). The different abiotic stress factors such as drought, light,
and Adamska, 2001; Yang et al., 2009; Xue et al., 2009). Other than
these functions, plant MTs have been also reported to regulate other
cellular processes such as cell growth and DNA damage repair (Cherian
and Kang, 2006). A study reported that the MTs may significantly
change the redox status and shows antioxidant property against both
hydroxyl and peroxyl radicals when people consume sweet potato
(Huang et al., 2014). Another study demonstrates that in in vivo con-
dition, MTs function as a zinc reservoir, which releases from MT, when
the levels of reactive nitrogen species (RNS) and ROS increase (Bell and
Vallee, 2009). Zinc dependent genes are involved in the regulation and
maintenance of stress tolerance mechanism in the plants (Calmak,
2000). This indicates the indirect regulation of tolerance mechanism by
MTs in plants. Plants have developed an elaborate antioxidant system to
combat ROS stress by increasing the level of low molecular weight
antioxidants (glutathione, ascorbate, MTs) and ROS scavenging

⁎
Correspondence to: Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, India.
E-mail address: i.sanyal@nbri.res.in (I. Sanyal).

https://doi.org/10.1016/j.ecoenv.2018.12.050
Received 28 July 2018; Received in revised form 13 December 2018; Accepted 16 December 2018
Available online 28 December 2018
0147-6513/ © 2018 Elsevier Inc. All rights reserved.
A.K. Dubey et al.
enzymes like superoxide dismutase (SOD), guaiacol peroxidase (POD),
ascorbate peroxidase (APX) and catalase (CAT) (Apel and Hirt, 2004).
Thus, above reports suggest that plants can survive under different
stress, such as drought, salinity and heavy metals by the enhancement
of several defense related mechanisms including MTs, although, the
role of MTs in scavenging ROS has still to be deciphered. A study re-
ported that MT deficiency in Arabidopsis affects Cu accumulation and
distribution in leaves and seeds (Benatti et al., 2014). Metallothionein
gene expression was strongly induced by Cu but to a lesser degree by Cd
and Zn in Arabidopsis, rice and in the metal hyper-accumulator Noccaea
caerulescens (Zhou and Goldsbrough, 1994; Hsieh et al., 1995; Guo
et al., 2003; Sancenón et al., 2004). Several studies described the ex-
pression of plant MTs in response to biotic and abiotic stress (Mekawy
et al., 2018; Sekhar et al., 2011).
The adverse environmental conditions such as drought, high salinity
and low temperature significantly affect the growth and productivity of
plants. In these conditions, drought is a major problem for agricultural
production in most parts of the world (Wang and Komatsu, 2017; Miller
et al., 2010; Wang et al., 2018). The exposure of drought stress, leads to
the inhibition of growth and productivity in plants through the al-
teration of biomolecular structures and metabolic process (Kashiwagi
et al., 2006; Dixit et al., 2017). The plants adapt to drought stress via
several modes: (1) escape: plant completes its life cycle before severe
water shortfall; (2) avoidance: plants develop deeper root system to get
water and reduces the number of stomata as well as leaf area; (3) os-
motic adjustment: plants improve their ability of osmotic adjustment
and increase tissue turgidity; (4) tolerance: plants increase their anti-
oxidant activity to scavenge ROS; (5) discarding: plants remove their
parts, which is exposed to the water deficit stress; (6) genetic mod-
ification: plant exposed to the long-term drought conditions, thrust
plants for the evolution of biochemical-physiological traits via genetic
mutation.
In this study, the chickpea metallothionein (CarMT) type 1
(LOC101494811, NCBI ref seq XM_004501707.1) based on its high
transcript level, studied by Molina et al. (2008) and also its high re-
lative expression in our study (Fig. 1B), under drought stress, has been
taken for exploring its role against drought stress in Arabidopsis
thaliana. The over-expressed lines were subjected to drought stress and
several drought stress-responsive biochemical and physiological para-
meters evaluated to examine the role of MT in tolerance response. The
possible role of MT in mitigating drought stress in plants is by the
modulation of the antioxidant system for scavenging ROS, thereby in-
creasing the physiological performances, relative water content and
stress-responsive amino acids. These findings show a specific protective
role of MT gene against drought stress and provide a genetic en-
gineering strategy, that may be helpful to improve tolerance against
drought stress. This work also contributes to a better understanding of
plant MTs and is the basis for further analysis of structure/function of
Fig. 1. Total transcript level of MT gene. (A) Relative expression of MT gene in transgenic Arabidopsis under drought stress, (B) Expression of MT gene in two
contrasting varieties of chickpea.
55
Ecotoxicology and Environmental Safety 171 (2019) 54–65
this protein during drought stress.
2. Materials and methods
2.1. Plant materials and experimental design
Two chickpea varieties (sensitive and tolerant) were taken to screen
the expression level of MT gene under drought stress. The aseptically
grown chickpea were inoculated in perforated cups fixed on plastic
support in a plastic tray that was hydroponically supplemented with
Hoagland nutrient solution. PEG (15% and 25%) was used to induce
drought stress in well grown seedlings of chickpea. Each tray had 24
seedlings of both the tolerant (A1–A4) and sensitive (B1–B4) varieties
and was supplemented with 15% and 25% PEG respectively. Each
treatment was repeated simultaneously in four trays, to minimize var-
iation amongst treatments. The control seedlings were grown in
Hoagland nutrient solution only. After seven days of treatment with
PEG, total RNA was isolated from the leaves of chickpea to analyze the
expression of MT gene using MT gene primers (Supplementary Table 1).
Chickpea actin gene was used as reference control. The seeds of Ara-
bidopsis thaliana Col-0 were sown in plastic containers filled with soil-
rite and irrigated with Hoagland's nutrient solution with EC value 2.1
dS m−1. It is one of the most popular solution for growing plants that
provides every nutrient necessary for plant growth and development for
a large variety of plant species. The containers were kept at 4 °C for 48 h
and then grown under 16 h light photoperiod of 150 µmol s−1 m−2
provided by cool white fluorescent light, at 22 ± 0.05 °C (In order to
mimic the ambient light conditions, which could be informative for
optimizing field growth and reproducibility in future experiments). The
Arabidopsis plants were irrigated with Hoagland's solution on alternate
days. When the plants reached 8–10 cm in height, the shoot tips were
clipped once, to favour the emergence of multiple secondary bolts in
the plants and were used for Agrobacterium-mediated genetic transfor-
mation (Clough and Bent, 1998).
The seeds of transgenic plants were sown in soilrite potting mix for
the evaluation of drought tolerance. Eight transgenic lines were
screened and selected for drought endurance. The wild-type and
transgenic lines were irrigated with nutrient solution for normal growth
and development. After growth of seedlings, they were subjected to
drought stress for seven days by water holding. Initial drought tolerance
was determined by growing the seeds in ½ MS plates supplemented
with 300 mM mannitol. All the genetic, physiological and biochemical
analysis was done in third generation transgenic lines of Arabidopsis,
abbreviated as T3-1 to T3–8 lines, wild-type control as WT and un-
treated control as C.
A.K. Dubey et al.
2.2. Construct preparation and Agrobacterium-mediated transformation
The cDNA containing an open reading frame of chickpea MT gene
(728 bp) was synthesized and cloned into the pUC19 vector at the SmaI
site. The pUC19 containing MT gene was isolated and transformed in
E.coli. The vector was digested with HindIII and SacI, and the gene
fragment was isolated and purified using the gel extraction kit
(Promega, USA) as per the manufacturer's instructions. This fragment
was ligated using DNA ligase into plant expression vector pBI121 to
derive pBI121MT driven by the constitutive CaMV35S promoter. The
pBI121MT construct was then transferred into Agrobacterium tumefa-
ciens GV3101 strain by freeze-thaw method (Jyothishwaran et al.,
2007) (Supplementary Fig. 3). The presence of the MT gene was con-
firmed by PCR with suitable primers (Supplementary Table 1.).
The Arabidopsis plants were inoculated with Agrobacterium tumefa-
ciens GV3101 strain harbouring the pBI121MT construct by floral dip
method (Clough and Bent, 1998). The uppermost part of the plants was
dipped for 30 s with gentle agitation in 100 ml solution of 5% (w/v)
sucrose with Agrobacterium tumefaciens cells at 0.8 A600 and 0.02% (v/
v) of the surfactant Silwet L-77 (Lehle Seeds, USA). Plants were covered
with a cloth along with a container containing water to maintain hu-
midity and kept for 24 h. After 24 h, the plants were kept till seed
setting, in the plant growth chamber (Adaptis 1000 PG, Conviron, Ca-
nada).
Putative transformants (T1) were selected based on their resistance
to kanamycin (50 mg L−1) incubated on ½ MS medium containing 2%
(w/v) sucrose and 0.8% (w/v) agar. Further, the presence of MT gene
was verified in the putative transformants by PCR with the genomic
DNA using MT gene-specific primers (Supplementary Fig. 4). The plants
were grown in the growth chamber up to the T3 generation to obtain
the homozygous transgenic lines. These T3 plants were analysed for
transgene expression and for the functional validation of the MT gene.
2.3. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
For the evaluation of the expression of MT gene, the total RNA was
isolated using Spectrum plant total RNA kit (Sigma Aldrich, USA) from
the leaves of Arabidopsis plants of the T3 generation. The RNA was
converted into cDNA using RevertAid First Strand cDNA Synthesis Kit
(Thermo Scientific, USA). This cDNA was used as a template for
quantification of the total transcript using 2x master mix SYBR® green
dye (Thermo Scientific, USA) in 7500 fast real-time PCR machine
(Applied Biosystems, USA). Arabidopsis actin gene was used as a re-
ference control. The relative expression of the gene was analysed using
the 2-^^CT method. The expression was calculated by the 2-^^CT method
(Livak and Schmittgen, 2001). The primer sequences are shown in
Supplementary Table 1.
2.4. Biochemical analysis of transgenic A. thaliana under drought stress
Total protein concentration in cell-free extracts was determined by
the dye-binding method of Bradford (1976) using bovine serum al-
bumin (BSA) as a standard protein. 10 μl of diluted protein sample was
mixed with 200 μl of Bradford dye (Sigma-Aldrich, USA) and the ab-
sorbance was measured at 595 nm.
Thiobarbituric acid reactive substances (TBARS) content was esti-
mated spectrophotometrically at 532 nm and 600 nm following the
method of Hodges et al. (1999), using 6.65% thiobarbituric acid, 0.01%
BHT mixed in 20% TCA and heated with the sample at 95 °C for 45 min.
H2O2 content was estimated by the method of Velikova et al. (2000).
The calculation was performed using ε = 0.155 M g−1 FW by deducting
the turbidity recorded at 600 nm from 532 nm. Ascorbic acid (µM g−1
FW) content was estimated spectrophotometrically at 525 nm following
Kampfenkel et al. (1995) by comparing it with the standard curve of L-
ascorbic acid (Sigma-Aldrich, USA). Reduced and total glutathione
(GSH and total GSH) were estimated according to the method of
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Ecotoxicology and Environmental Safety 171 (2019) 54–65
Anderson (1985) by estimating the stoichiometric formation of 5-thio-
2-nitrobenzoic acid (TNB) at 412 nm and comparing with a standard
curve prepared with GSH (Sigma-Aldrich, USA).
For the analysis of enzyme activity, fresh leaf samples (300 mg)
were ground using liquid N2 in a chilled mortar and pestle and ex-
tracted with 3 ml of 100 mM potassium phosphate buffer (pH 7.5)
containing 1 mM EDTA and 1% (w/v) polyvinylpyrrolidone (PVP). The
homogenate was centrifuged at 12,000 rpm for 15 min. Superoxide
dismutase (SOD) (EC 1.15.1.1) activity was measured spectro-
photometrically at 560 nm following Beauchamp and Fridovich (1971)
and presented as U mg−1 protein, where 1 U of SOD activity is the
amount of protein required to inhibit 50% of initial reduction of nitro
blue tetrazolium (NBT) under the light. Ascorbate peroxidase (APX) (EC
1.11.1.11) activity was measured following Nakano and Asada (1981)
using ε = 2.8 mmol−1cm−1 and enzyme activity was expressed as µM
of ascorbate oxidised min−1 mg−1 protein. Catalase (CAT) (EC
1.11.1.6) activity was measured by the method of Chandlee and
Scandalios (1984) and expressed as mmol min−1 mg−1 protein.
Guaiacol peroxidase (POD) (EC 1.11.1.7) activity was measured at
470 nm following Kato and Shimizu (1987) using ε = 26.6 mmol−1
cm−1 and expressed as µM of guaiacol oxidised min−1 mg−1 protein.
Glutathione reductase (GR) (EC 1.6.4.2) activity was assayed following
Smith and Johnson (1988) and represented as U mg−1 protein, where
1U is the conversion of 1 mM of oxidised glutathione (GSSG) min−1
into reduced glutathione (GSH). Monodehydroascorbate reductase
(MDHAR; EC 1.6.5.4) activity was assayed following Vanacker et al.
(1998) by monitoring the formation of monodehydroascorbate at
340 nm (1 mM of ascorbate oxidised min−1) and represented as mM
mg−1 protein. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) activity
was measured by Doulis et al. (1997) at 265 nm using
ε = 7.0 mmol−1cm−1 and represented as µmolmg−1 protein. Glu-
tathione peroxidase (GPX) activity was measured by Takeda et al.
(1993) in a total volume of a 1 ml reaction mixture containing 100 mM
Tris-HCl (pH 7.5), 1 mM GSH, 0.4 mM NADPH, 0.2 mM H2O2 and 1 unit
of GR.
The depletion of NADPH was monitored at 340 nm, which reduces
H2O2 to H2O. Glutathione S-transferase (GST; EC 2.5.1.13) activity was
measured according to Habig et al. (1974) using ε = 9.6 mmol−1cm−1
of the product and expressed as µM of CDNB (1-Chloro-2, 4-dini-
trobenzene) conjugated mg−1 protein. The depletion of NADPH was
monitored at 340 nm, which reduces H2O2 to H2O. Glutaredoxin ac-
tivity was determined by measuring the reduction of 2-hydroxyethyl
disulfide (HED) in the presence of NADPH and glutathione reductase.
The decrease in NADPH was monitored at 340 nm using a molar ex-
tinction coefficient of 6200 M−1 cm−1. The reaction mixture contains
1 mM GSH, 0.2 mM NADPH, 2 mM EDTA, 0.1 mg ml−1 bovine serum
albumin, and 6 µg ml−1 yeast glutathione reductase, prepared in
100 mM Tris-HCl, pH 8.0, to 500 μl of this mixture, HED was added at a
final concentration of 0.75 mM. The decrease in absorbance at 340 nm
was recorded using a UV/Vis spectrophotometer (Perkin Elmer, USA).
Total free cysteine content was estimated spectrophotometrically
according to Gaitonde (1967) by recording the absorbance at 560 nm.
Proline was determined according to Bates et al. (1973), based on
proline's reaction with ninhydrin. For proline determination, a 1:1:1
solution of proline, ninhydrin and glacial acetic acid was incubated at
100 °C for 1 h. The chromophore was extracted with 4 ml toluene, and
its absorbance at 520 nm was determined in a spectrophotometer
(Thermo Fisher Scientific, USA).
2.5. Measurement of physiological performance
Both the WT and transgenic lines (T3-1 to T3–8) were subjected to
simultaneous leaf gas exchange and chlorophyll fluorescence mea-
surements [Photosynthesis rate (A; µmol m−2 s−1), Stomatal con-
ductance (G; mol H2O m−2 s−1), Leaf transpiration (E; mmol H2O m−2
s−1), Water use efficiency (WUE; µmolCO2 mmolH2O−1), Intercellular
A.K. Dubey et al.
to ambient CO2 conc. ratio (Ci/Ca), Variable to maximum fluorescence
ratio (Fv/Fm), Photochemical quenching (qP), Non- photochemical
quenching (qN) and Electron transport rate (ETR; µmol e- m−2 s−1)] by
an open flow gas exchange system (Li-Cor 6400XT; Li-Cor, Inc., Lincoln,
NE, USA). The measurements were undertaken on fully expanded, un-
damaged leaves following drought stress for seven days. The air flow
rate, relative humidity, Tleaf, VPD, and CO2 concentration was kept
constant for all the treatments, at 55%, 300 µmol s−1, 25 °C, 1.5 KPa
and 400 µmol mol−1 respectively. The plantlets were subjected to sa-
turating photosynthetic photon flux density (PPFD) of 1500 µmol
m−2s−1 (provided by artificial LED light source Li-Cor 6400–02B).
2.6. Determination of cell membrane injury (electrolyte leakage/relative
conductivity)
At the last day of drought treatment, the leaf pieces were collected
and washed three times in 10 ml of deionised water and then immersed
in 10 ml of deionised water and kept for 3 h at 25 °C. Electrical con-
ductivity was measured with EC meter, and then the leaf tissues were
killed by boiling for 15 min at 70 °C, cooled to 25 °C, and the electrical
conductivity was measured for the second time. Membrane injury was
evaluated as the percentage injury index following the equation of
Bandurska (2000).
I = C1/C2 × 100%
where C1 and C2 represent conductivity measurements of control
samples before and after boiling, respectively.
2.7. Determination of relative water content (RWC)
For determination of RWC, from each plant, a leaf was cut and
weighed to determine the fresh weight and positioned with the cut end
in tap water in a closed vessel. After 14 h incubation at RT, turgid
weight (TW) was determined (Turner, 1981; Lafitte, 2002). Leaf sam-
ples were then dried for 72 h at 70 °C to determine the dry weight (DW).
Relative water content was calculated using the equation:
RWC = (FW − DW) × 100/(TW − DW)
2.8. Evaluation of drought tolerance in A. thaliana transformed with MT
gene
Drought tolerance in transgenic plants harbouring the MT gene were
evaluated by germinating the seeds of WT and transgenic seeds on ½
MS medium containing 2% (w/v) sucrose and 0.8% (w/v) agar with or
without 300 mM mannitol. Seeds were surface sterilised with 1.5% (v/
v) sodium hypochlorite solution followed by washing with 4–5 times in
sterilised water. Approximately, 100 seeds were spread on ½ MS media
and kept at 4 °C for two days for stratification. After stratification, the
plates containing seeds were transferred into the plant growth chamber
under cool white light at 23 °C for 3–4 days to observe the germination
rate in WT, and transgenic seeds on mannitol-supplemented media.
Root length under drought stress was determined by transferring the
four-day-old plantlets into agar plates containing mannitol. For growth
parameter measurements, root length and fresh weight was determined.
2.9. Statistical analysis
All the obtained values are the average of three replicates. The
values are represented as a percentage increase or decrease with respect
to the respective values in WT or otherwise mentioned. The data were
subjected to Duncan's Multiple Range Test (DMRT) for the analysis of
significant difference between the means (p < 0.05). SD values were
calculated using the mean of the replicates.
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Ecotoxicology and Environmental Safety 171 (2019) 54–65
3. Results
3.1. Screening of transgenic lines
The genomic DNA of wild-type and transgenic Arabidopsis plants
were isolated and used for PCR analysis using MT gene specific primers.
The transformants showed amplification of the MT gene while no am-
plification was observed in wild-type plants (Supplementary Fig. 2).
3.2. Transgenic lines showed higher transcript level of MT gene
Total transcript level was analysed by the qRT-PCR using a gene
specific set of primers. Arabidopsis actin gene was used as an internal
control to normalise the mRNA level. All over-expressing transgenic
lines showed higher transcript level than WT plant against drought
stress. The increase in transcript level was observed in all the transgenic
lines, and it ranges from 9.37 to 30.25 fold change higher as compared
to the WT. The highest transcript level was observed in T3–6 line which
was 30 fold change higher than WT plant (Fig. 1A).
3.3. Effect of over-expression of the MT gene on plant growth parameters
Germination of WT seeds was significantly inhibited on mannitol-
supplemented plates while germination rate and survival efficiency
were increased in transgenic seeds germinated on ½ MS plates sup-
plemented with mannitol (Supplementary Fig. 1). The seeds of WT and
transgenic lines were surface sterilised and spread on ½ MS medium
supplemented with 300 mM mannitol. After a certain period, the ger-
mination was observed and found that the transgenic seeds were
growing faster than WT seeds. The germinated seedlings were trans-
ferred on Petri plates containing same concentration of mannitol, in-
cubated vertically, in which root growth was observed and measured
after a certain time period. The roots of transgenic plants were longer
than those of the WT Arabidopsis seedlings and increased from 2.5 cm in
WT, to up to 6.5 cm in T3–6 line. Similarly, the transgenic plants
showed higher biomass as compared to WT seedlings, increased from
2.8 mg in WT, to up to 10 mg in T3-3 line (Fig. 2A, B, D). The size of
siliques in the transgenic Arabidopsis was greater than WT plant grown
under drought stress (Supplementary Fig. 2).
3.4. Transgenic plants showed lower TBARS, electrolyte leakage and higher
antioxidant levels
The response of drought stress in transgenic and WT Arabidopsis was
examined by the change in biochemical parameters. In our study, the
total content of protein was higher in transgenic lines as compared to
WT plants. The highest content was measured in T3-2 and T3–4 lines
under drought stress, which were 8.30 and 8.15 mg g−1 FW respec-
tively i.e. 89% and 85% respectively higher than the WT plants
(Table 1).
The stress marker, thiobarbituric acid reactive substance (TBARS)
was increased in WT plants (11.87 µM/g FW), while it was decreased in
all lines but significant decrease was observed in T3-1, T3–4, T3–7 and
T3–8 lines of transgenic Arabidopsis transformed with MT gene under
drought stress (Table 1). The maximum decrease in TBARS level was
observed in the transgenic line T3–7 (9.15 µM/g FW). Unexpectedly,
the H2O2 level was increased in all the transgenic lines under drought
stress (Table 1).
Electrolyte leakage is the signature of plasmolysis which denotes the
permeability of ions during stress. In this study, WT plants showed
higher percentage of electrolyte leakage while all transgenic lines
showed low percentage of electrolyte leakage. The significant decrease
in EL value was observed in T3-1, T3-2, T3-3, T3–4, T3–5 and T3–8
lines (Fig. 3).
Under oxidative stress conditions, plant cells shift from reduced
towards the oxidised environment. To re-maintain the reducing
A.K. Dubey et al. Ecotoxicology and Environmental Safety 171 (2019) 54–65

Fig. 2. Morphological measurements of the control, wild-type and transgenic plants under drought stress. (A) Root morphology of transgenic, WT and control on
300 mM mannitol, (B) Root length of transgenic, WT and control seedlings in cm, (C) Biomass of individual WT, control and transgenic seedlings in mg.

environment, plants synthesize reducing agents such as ASc and GSH.
In present study, the ratio of ASc/DHA was enhanced in most of the
transgenic plants as compared to WT (Table 1). The highest increased
ratio (67%) of ASc/DHA was observed in T3–5 plants which was very
low in WT during drought stress. Similar to the trend of ASc/DHA, the
ratio of GSH/GSSH was decreased in WT, while, it was increased in
transgenic plants during drought stress (Table 1). The maximum in-
creased ratio (243%) was observed in T3–7 line, which was higher as
compared to WT.
3.5. Effect on drought responsive amino acids
In our study, the total content of proline amino acid was increased
in all the transgenic lines as compared to WT (Fig. 4A). The highest
content of proline (~5 fold) was observed in T3–6 line. Similarly, the
content of cysteine was also observed to be increased in all the trans-
genic lines (Fig. 4B). The maximum increase in cysteine content (~3
fold) was observed in T3-1 line.
3.6. Increased activities of antioxidant enzymes in transgenic lines against
drought stress
Ascorbate peroxidase (APX) is a key enzyme which plays a major
role in catalysing the conversion of H2O2 into H2O, using ascorbate as a
specific electron donor, particularly in the chloroplast. In this study, the
APX activity was enhanced in transgenic Arabidopsis, while, the activity
was diminished in WT plants during drought stress. The highest APX
activity was shown by T3-3 and T3–8 transgenic lines, which were
487% and 488%, respectively, as compared to WT Arabidopsis plants
(Fig. 5A). Similarly, guaiacol peroxidase (POD) activity was found to be
very high in transgenic plants (Fig. 5B). The highest activity was ob-
served in T3–8 transgenic line which was significantly greater (135%)
(p < 0.05) than WT plant. Similarly, the GPX activity was significantly
increased in most of the transgenic Arabidopsis lines, under drought
stress as compared to WT (Fig. 5C). The maximum increase in GPX
activity (134%) was observed in T3–8 line. GR activity was found to be
enhanced against drought stress in all the transgenic lines as compared

Table 1
Different level of stress markers TBARS, H2O2, antioxidant molecules ASc/DHA and GSH/GSSH ratio and total protein content in control, WT and transgenic plants
under drought stress. Significance of the mean values have been compared for each parameter separately (Duncan's test, p < 0.05) where bars marked with same
letters are not significantly different.
Arabidopsis plants TBARS (µM g−1 FW) H2O2 (nM g−1 FW) ASc/DHA ratio GSH/GSSG ratio Protein (mg g-1 FW)

C 8.86 ± 0.28a 227.92 ± 40.78a 0.07 ± 41.78a 1.08 ± 41.78a 7.92 ± 41.78a
WT 11.86 ± 0.39b 237.92 ± 41.78a 0.09 ± 0.01a 1.26 ± 0.07a 4.40 ± 0.01a
T3–1 9.81 ± 1.26a 267.68 ± 38.67a 0.11 ± 0.00b 2.73 ± 0.84b 4.71 ± 0.12a
T3–2 10.29 ± 1.22ab 293.09 ± 29.39a 0.10 ± 0.00ab 2.18 ± 0.43b 8.30 ± 0.47c
T3–3 10.64 ± 0.25ab 220.75 ± 1.61a 0.12 ± 0.03b 2.87 ± 0.46b 5.82 ± 0.14ab
T3–4 9.17 ± 0.76a 344.42 ± 42.69b 0.13 ± 0.01b 3.23 ± 0.28c 8.15 ± 0.20c
T3–5 10.53 ± 0.46ab 238.71 ± 42.77a 0.15 ± 0.07c 2.97 ± 0.39b 5.59 ± 0.05ab
T3–6 10.56 ± 0.46ab 340.75 ± 3.23b 0.14 ± 0.01c 3.19 ± 0.91c 6.72 ± 0.02b
T3–7 9.15 ± 1.29a 314.40 ± 68.34b 0.12 ± 0.00b 4.32 ± 0.31d 5.95 ± 0.02ab
T3–8 9.31 ± 0.22a 278.51 ± 11.91a 0.13 ± 0.01b 4.22 ± 0.49d 5.56 ± 0.02ab

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Fig. 3. (A) Percentage of electrolyte leakage in wild-type and transgenic plants under drought stress, (B) Percentage of relative water content in transgenic and wild-
type plants under drought stress.
to WT (Fig. 5D). Similar to GR, the activity of GRX was increased many
folds in the transgenic lines (Fig. 5E). The highest GRX activity (186%)
was observed in T3-1 transgenic line. Also, the activity of SOD, CAT and
GST was enhanced in transgenic lines (Fig. 5F, G, H). On the other
hand, the activity of MDHAR was observed to be enhanced only in three
transgenic lines i.e. T3–6, T3–7 and T3–8 transformed with MT gene as
compared to WT (Fig. 5I). However, the most of the transgenic lines
showed higher activity of DHAR (Fig. 5J). Overall, the results suggested
that the antioxidant enzymes showed enhanced activity in the trans-
genic lines (APX, GPX, CAT, POD, GRX, GST, SOD, GR and DHAR) to
counter the drought stress.
3.7. Effect on physiological performances and relative water content
Physiological performance of Arabidopsis plants transformed with
MT gene was evaluated for drought stress endurance. Photosynthetic
assimilation rate per unit leaf area (A; µmol m−2 s−1) was observed
highest (6.13 ± 0.19) in T3-1 line while lowest (2.77 ± 0.16) car-
boxylation rate was observed in WT (Fig. 6B). The MT gene was seen to
promote drought enduring capabilities in T3-1 and T3–5 lines, which
was reflected in high instantaneous and intrinsic water use efficiencies
in T3–5 (0.33), T3-1 (0.30) lines (Fig. 6A). The MT gene undisputedly
enhanced carboxylation rates in all the transgenic plants (T3-1 to T3–8)
which were corroborated by high stomatal conductance and leaf tran-
spiration values as compared to WT which showed lowest values
(Fig. 6C, H). In T3-2, T3-3, T3–4, T3–6 and T3–7 lines, although the MT
gene caused an incremental upregulation in carboxylation rates and
Fig. 4. (A) Total content of proline amino acid in control, wild-type and transgenic Arabidopsis thaliana under drought stress. (B) Total content of cysteine amino acid
in control, wild-type and transgenic Arabidopsis thaliana under drought stress. Values marked with same alphabets are not significantly different (DMRT, p < 0.05).
All the values are means of three replicates ± SD.
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Ecotoxicology and Environmental Safety 171 (2019) 54–65
also higher WUE in few transgenic lines (T3-1 and T3–5 lines) and
decreased in remaining lines. Similarly, T3-3 type showed highest ETR
values (82.65 ± 4.87 µmole m−2 s−1) which was due to excessive
excitation of the two photosystems leading to heat dissipation by non-
photosynthetic quenching pathway which were further reflected in
highest qN (0.79 ± 0.04) and low photosynthetic values (Fig. 6F, G).
Fv’/Fm’ values varied from 0.53 ± 0.02 (T3-2) to 0.64 ± 0.03 (T3–7)
among all the Arabidopsis plants (Fig. 6D).
The relative water content was measured in WT and transgenic lines
under drought stress. All the transgenic lines showed higher relative
water content as compared to WT Arabidopsis plants and significant
RWC was observed in T3-3, T3–4, T3–5 and T3–6 lines (Fig. 3). The
highest relative water content was observed in T3–5 line which was
91% higher as compared to WT plants.
4. Discussion
Many studies revealed that the expression of MT gene is induced
during drought stress in addition to its role in metal tolerance and
homeostasis (Molina et al., 2008; Yang et al., 2009; Jin et al., 2017). In
our study, the high expression of MT gene under drought stress in tol-
erant cultivar and low expression in sensitive cultivar shows the in-
volvement of MT gene in amelioration of drought stress, which is
supported by a similar study in two rice cultivars by Shankar et al.
(2016). This gene was over-expressed in Arabidopsis thaliana and
drought related parameters were analysed, which showed positive re-
sponse during drought stress. Many studies showed the role of MTs in
A.K. Dubey et al. Ecotoxicology and Environmental Safety 171 (2019) 54–65

Fig. 5. Antioxidant enzyme activities in control, transgenic and wild-type Arabidopsis plants under drought stress. (A) APX (μmol g−1 P), (B) POD (μmol g−1 P), (C)
GPX (μmol g−1 P), (D) GR (μmol g−1 P), (E) GRX (μmol g−1 P), (F) SOD (μmol g−1 P), (G) CAT (μmol g−1 P), (H) GST (μmol g−1 P), (I) MDHAR (μmol g−1 P) and (J)
DHAR (μmol g−1 P). In units, P denotes total protein. Significance of the mean values have been compared for each parameter separately (Duncan's test, p < 0.05)
where bars marked with same letters are not significantly different.

amelioration of metal toxicity, but its role against drought stress, still lines under drought stress. Higher expression of the MT gene was ob-
remains unexplored. In a study by Molina et al. (2008), MT gene was served in the transgenic lines of Arabidopsis as compared to the wild-
found to be upregulated in chickpea under drought stress. Our study type control due to the site of integration of the transgene in the
also showed the higher expression of MT gene in Arabidopsis transgenic genome. Higher relative expression of the MT gene, leads to the better

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A.K. Dubey et al. Ecotoxicology and Environmental Safety 171 (2019) 54–65

Fig. 6. Physiological performance in transgenic lines and wild-type plants under drought stress. (A) Water use efficiency (WUE; µmolCO2 mmolH2O−1), (B)
Photosynthesis rate (A; µmol m−2 s−1), (C) Stomatal conductance (G; mol H2O m−2 s−1), (D) Variable to maximum fluorescence ration (FV/Fm), (E) Photochemical
quenching (qP), (F) Non-photochemical quenching (qN), (G) Electron transport rate (ETR; µmol e- m−2 s−1), (H) Leaf transpiration (E; mmol H2O m−2 s−1), (I)
Intercellular to ambient CO2 ratio (Ci/Ca).

61
A.K. Dubey et al.
protection of plants under drought stress that is supported by the dif-
ferent physiological and biochemical parameters. The higher expression
of MT under drought stress was also corroborated with a study by Yang
et al. (2009), where, they have shown its involvement in combating
drought stress. In another study, higher expression of MT gene under
moderate drought stress was observed in wheat (Moloudi et al., 2013).
The seed germination and survival efficiency of transgenic lines on
300 mM mannitol-supplemented plates were greater than WT plants,
which shows better tolerance in transgenic lines against drought stress.
The larger root length and greater biomass of transgenic lines also
supports their improved tolerance in drought stress. The larger siliques
of transgenic lines compared to WT also supports its enhanced pro-
ductivity against drought stress. This is supported by a study of Yang
et al. (2009), where similar trends were observed in rice seedlings. It
has also been reported that plant MTs and other metalloproteinases are
involved in root development and other developmental processes in
plant (Mir et al., 2004; Liu et al., 2017). In another study in rice, MT has
been shown to induce cell proliferation and reduce cell death during Cu
and Cd stress (Zhang et al., 2017).
Amino acids play an important role in drought stress which is re-
ported by various studies (Rai, 2002; Nanjo et al., 1999; Yang et al.,
2000; Good and Zaplachinski, 1994). In present study, the amino acids
i.e. proline and cysteine was observed to be enhanced in transgenic
lines as compared to WT plants, which shows the enhanced defence
mechanism against drought stress. These are stress responsive amino
acids, where proline acts as an osmolyte, while cysteine contains thiol
group for chelation of toxic metals, which significantly participate in
abiotic stress tolerance (Ashraf and Foolad, 2007; Alvarez et al., 2012).
Abiotic stress induces over-production of ROS which causes the
degradation of lipid, nucleic acid and protein molecules, leading to the
cell death. TBARS is a main stress marker which increases during stress
condition (Dubey et al., 2016). In our study, the level of TBARS was
found to be increased during drought stress in WT, while, it was re-
duced in the transgenic lines and untreated control. The reduced level
of TBARS in transgenic lines shows less toxicity under drought stress.
The electrolyte leakage is the result of higher lipid peroxidation during
stress conditions (Fu and Huang, 2001; Niedzwiedz-Siegien et al., 2004;
Blokhina et al., 2003). During this study, MT gene was seen to reduce
the electrolyte leakage in the Arabidopsis plants transformed with MT
gene. The other stress marker and ROS i.e. H2O2 was found to be higher
in transgenic lines. The higher level of H2O2 might be due to its sig-
naling property for defense system against drought stress. H2O2 acts as
a signal molecule and helps MTs in ROS scavenging, where, H2O2 is
shown to enhance the expression of MT gene for providing tolerance
against Cu and Cd stress in rice seedlings (Zhang et al., 2017). The
regulation of H2O2 mediated gene expression is the cross-link between
the variety of stress stimuli and different signaling pathways (Mylona
and Polidoros, 2010).
Drought stress causes the generation of ROS which is overcome by
various antioxidant molecules and antioxidant enzymes (Akashi et al.,
2004). During this study, the antioxidant molecules ASc/DHA ratio was
enhanced in all transgenic lines as compared to WT plants and un-
treated control that shows the strong defence system in transgenic
plants to combat the drought stress. Similarly, the other important
antioxidant molecules i.e. GSH/GSSG ratio was also observed to be
enhanced in all the transgenic lines as compared to WT and untreated
plants, that shows the strong defence system dependent upon GSH
molecule. This ratio is very important for reducing environment of the
cells during oxidative stress. This is corroborated with the study by
Nahar et al. (2015), where, they have shown the role of GSH in drought
stress. Ascorbate and GSH are important reducing agents in the cell,
which are actively involved in the cellular homeostasis and act as a co-
substrate for antioxidant enzymes (Foyer and Noctor, 2005). The an-
tioxidant enzymes are the important part of the plant defense system
which actively participates in removal of ROS during oxidative stress
(Takahashi et al., 2013). In present study, during drought stress, the
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Ecotoxicology and Environmental Safety 171 (2019) 54–65
transgenic lines showed enhanced activities of antioxidant enzymes
(APX, GPX, POD, GRX, SOD, GR, MDHAR, GST and CAT) which coin-
cided with the reduced level of TBARS and increased level of GSH/
GSSG ratio in the plants. Higher activities of antioxidant enzymes in
plant signify the tolerance response against stress (Li et al., 2016). In
our study, the APX and GPX activities were enhanced in all transgenic
lines under drought stress which shows the protective role of MT as an
inducer of APX and GPX enzymes. The transgenic line T3-1 showed
higher activity of all antioxidant enzymes as compared to WT. A similar
study was reported by Yang et al. (2009), where, APX, POD and CAT
activities were enhanced in transgenic rice under drought stress. An-
other important antioxidant enzyme i.e., SOD (first line of defence
against ROS) was also enhanced in the transgenic lines, which might be
due to higher expression of MT gene in Arabidopsis. The overexpression
of different metabolic pathway genes also coordinate with the potential
antioxidant enzymes such as CAT, APX, GPX, POD and SOD to eliminate
the elevated level of ROS (Dutta Gupta, 2010). In a study, the trans-
genic Arabidopsis plants suppressing CAT1-, APX1- and Cu/ZnSOD
genes under control conditions exhibited induction of different chlor-
oplastic and nuclear-encoded genes (Gadjev et al., 2006).
MTs act as a scavenger of ROS, either independent of SOD or act as
an activator of SOD by supplying metal ions (Koh and Kim, 2001).
Glutaredoxin enzyme is also an important antioxidant enzyme which
catalyzes the reversible glutathionylation of protein during adverse
condition to protect cellular proteins from damage by ROS (Klatt and
Lamas, 2000). In this study, the GRX activity was enhanced in MT
transformed transgenic lines which show the protective role of GRX
from ROS against drought stress as in a similar study in rice by Verma
et al. (2016). The GRX and antioxidant system supporting enzyme i.e.
Glutathione reductase (GR) catalyzes the reduction of oxidised GSH by
the NADPH and this reduced GSH has a wider function in cell's meta-
bolism (Lillig et al., 2008). During this study, all transgenic lines
showed higher activity of GR which is supported by the increased ratio
of GSH/GSSG. Similar study was made by Verma et al. (2016), where,
they have shown the enhanced activity of GR against arsenic stress in
rice. The other antioxidant enzymes like GST and MDHAR activities
were also enhanced that shows the strong antioxidant defence system
against drought stress in transgenic lines as compared to WT plants.
Similarly, enhanced antioxidant activities were also reported against
drought stress in white clover (Li et al., 2016).
Drought causes negative effect on physiological performances of the
plants, leading to lower photosynthesis rate, WUE, ETR and Fv/Fm
(Singh et al., 2014; Anjum et al., 2011). In this study, photosynthesis
rate was observed to be inhibited in WT while transgenic lines showed
increased rate of photosynthesis which might be due to the over-
expression of MT gene. Water use efficiency (WUE) is the relationship
between water use by the plants and total yield of the crop. Water
limitations can impose drought stress which is most important factor
limiting crop yield worldwide and it can be improved through drought
avoidance or drought tolerance (Price et al., 2002). During drought,
crop yield is determined by the rate of WUE which affects the tran-
spiration rate and stomatal conductance of the plants (Polley, 2002).
During this study, MT gene over-expression causes a significant increase
in stomatal conductance (G) and leaf transpiration rate (E), resulted
into high rate of photosynthesis (A) with slight increase in WUE in some
transgenic lines, while it was decreased or unaffected in the remaining
lines with recovered biomass. Recovered biomass with lower WUE
shows tolerance in transgenic lines which is supported by several stu-
dies (Edwards et al., 2012; Kørup et al., 2018). In some studies, it is also
reported that WUE is negatively correlated with the biomass production
and grain yield (Condon et al., 1987; Condon and Richards, 1993;
Fischer et al., 1998). A similar study was reported by Karaba et al.
(2007), where, they have shown the role of HRD gene in modulation of
WUE, stomatal conductance, leaf transpiration during drought stress.
The photosynthetic efficiency of the transgenic lines harbouring the MT
gene was higher as compared to WT. During drought stress, the leaf
A.K. Dubey et al.
water content loss reduces due to stomatal closure, CO2 supply lim-
itation and photosynthetic rate, to an extent of 60–80% (Marchese
et al., 2010; Carmo-Silva et al., 2009; Singh et al., 2014). In this study
also, the WT plants showed lower rate of photosynthesis while all the
transgenic lines exhibited higher rate of photosynthesis under drought
stress. However, no significant change was observed in the intercellular
to ambient CO2 ratio (Ci/Ca) in the transgenic lines. Transgenic lines
showed higher ETR values which were due to excessive excitation of the
two photosystems leading to heat dissipation by non-photosynthetic
quenching pathway. NPQ is the protection mechanism from ROS during
abiotic stress (de Bianchi et al., 2010; Murchie and Niyogi, 2011). Thus,
the increase in NPQ may represent an anticipatory response activated to
alleviate high light stress or drought stress. In this study, the transgenic
lines did not show significant change in NPQ but photochemical
quenching (qP) was higher in all transgenic lines as compared to WT.
This shows the better efficiency of photosystem II reaction centre of the
transgenic lines as compared to WT, during drought stress. The lower
NPQ and higher qP shows the minimum heat dissipation and maximum
utilization of light for photosynthesis in transgenic lines, under drought
stress, which, is also supported by increased rate of photosynthesis in
the transgenic lines. Higher efficiency of photosynthesis is also sup-
ported by unchanged Fv/Fm ratio in transgenic lines against drought
stress.
Relative water content is the most appropriate measurement of
drought stress in plant leaves. In many plant species, the relative water
content ranges from 80% to 90% and decreased to 40% in dying leaves
(Gonzalez and Gonzalez-Vilar, 2001; Schlemmer et al., 2005). In this
study, WT contained 62% RWC, while all transgenic lines showed
higher RWC even significantly higher in some transgenic lines, which
shows the tolerance improvement. This is in agreement with the study
of Jungklang and Saengnil (2012) who reported the ability of paclo-
butrazol to maintain leaf water content. Here the MT gene appears to
increase water content in drought-stressed Arabidopsis plants.
5. Conclusions
This study has attempted to explore the role of MT gene against
drought stress which is not yet reported, clearly in terms of physiolo-
gical and biochemical aspects. This study reveals that the transgenic
Arabidopsis plants transformed with MT gene enhances the defence
mechanism to overcome the ROS level, generated during drought stress.
In defence system, enhanced enzymatic (APX, GPX, SOD, POD, GRX,
CAT, GST, MDHAR and GR) and non-enzymatic (GSH and ASc) anti-
oxidants, are attributed to the MT gene-mediated tolerance mechanism
in Arabidopsis plants against drought stress. Simultaneously, the re-
duced TBARS level and electrolyte leakage with higher relative water
content and recovered physiological performances show improvement
in drought tolerance. Furthermore, higher expression of the MT gene in
the transgenic lines of Arabidopsis as compared to the wild-type control
leads to the better protection of plants under drought stress which is
supported by results obtained during the study of the different phy-
siological and biochemical parameters. Expression of the chickpea MT
gene can therefore be used to develop drought-tolerant transgenic
chickpea by over-expressing it in the drought-sensitive cultivars. Thus,
these findings show a protective role of the MT gene against drought
stress apart from its role in metal homeostasis by enhancing anti-
oxidants, drought responsive amino acids and recovery of physiological
variables, provides a genetic engineering strategy to improve drought
tolerance.
Acknowledgements
The authors are thankful to Director, CSIR-NBRI, Lucknow for the
infrastructural support to carry out the research. AKD acknowledges
CSIR, New Delhi for research fellowship.
63
Ecotoxicology and Environmental Safety 171 (2019) 54–65
Funding
The work and fellowship are supported by a research grant (BSC
0204) provided by CSIR, New Delhi, India.
Disclosure
This is to declare that the authors have no conflict of interest re-
garding authorship.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2018.12.050.
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