Journal of Ravishankar University–B, 34 (1), 69-79 (2021)
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Impact of Melatonin on Growth and Antioxidant Activity of Cicer arietinum L. Grown under Arsenic
Stress
Deepali Nagre, Roseline Xalxo, Vibhuti Chandrakar, S. Keshavkant
School
of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur-492010, C.G.,
India
*Corresponding author: skeshavkant@gmail.com
[Received: 10 March 2021; Revised: 7 June; Accepted: 11 June 2021]
Abstract. The ability of melatonin
to regulate number of physiological and biochemical processes under different
environmental stresses has been widely studied in plants. So, this
investigation was done to study the protective roles of melatonin on Cicer arietinum L. grown under arsenic
stress. Subjecting Cicer arietinum L.
seeds to arsenic stress caused significant decreases in germination percentage,
radicle growth, biomass accumulation, protein content and activities of
antioxidant enzymes. On the other hand, melatonin treatment significantly
increased growth parameters and protein quantity via improving antioxidant enzyme systems as compared with their
corresponding untreated controls.
Key words: Antioxidant
enzymes; Arsenic; Melatonin; Oxidative Stress.
Introduction
Arsenic
(As) is a non-essential metalloid, ubiquitously distributed in the water and
soil (Mirza et al. 2014). In the agricultural field, presence of As more than
the permissible limit (20 mg kg−1 soil) is injurious for the
vegetation of that area (Panda et al. 2010). In the soil, As subsists in its
four oxidation states, of which (+III) and (+V) are inorganic, and more deadly
forms, whereas; (-III) and (0) are organic as well as less lethal (Finnegan and
Chen 2012). Moreover, among all the inorganic forms, arsenite (AsIII)
is approximately 100-fold more soluble, mobile and cytotoxic in nature than the
arsenate (AsV) (Nath et al. 2014).The impact of irrigating the
agricultural field with high As contaminated water has pinched immense
attention due to transfer of As to the food chain via
groundwater-soil-plant system (Chandrakar et al. 2018). The bioaccumulation of
As in crop plants is potentially harmful to all the communities, and this is of
pronounced environmental alarm as As is known to be a carcinogen and a powerful
co-mutagen (Patra et al. 2004). Arsenate is an analogue of phosphate and thus obstructs
with essential cellular processes such as oxidative phosphorylation and ATP
synthesis, whereas the toxicity of AsIII is due to its propensity to
bind to -SH assemblies, with consequent damaging effects on general protein
functioning (Pandey et al. 2018).
Acquaintance of crop
plants to As outcomes in a range of adversative effects that comprises a number
of physiological disarrays including reduced/ inhibited germination, growth and
development depending upon level of As, and species contaminated with As (Chandrakar
et al. 2016). Exposure of crop plants to As upshots in abandoned production of
reactive oxygen species (ROS), and is a potential basis for oxidative damage to
important biomolecules like lipids, proteins and nucleic acids (Meharg and
Hartley-Whitaker 2002; Chandrakar et al. 2016). For usual growth, As-imposed
ROS must be quenched in the plant cells.
To keep themselves protected from the hazardous effects
of these reactive oxygen intermediates, plants are equipped with antioxidant
defense systems (Gill and Tuteja 2010; Chandrakar et al. 2017; Yadu et al.
2017). This defense system gets induced in order to protect the plants from several
stresses (Chandrakar et al. 2017; Yadu et al. 2017; Xalxo and Keshavkant 2019).
The antioxidant defense system comprises of enzymatic and non-enzymatic
components. Enzymatic agents includes superoxide dismutase (SOD), catalase (CAT),
ascorbate peroxidase (APX), guaiacol peroxidase (POD), glutathione reductase, etc., however; ascorbic acid, glutathione,
proline, glycinebetaine, phenolics, alkaloids, non-protein amino acids,
α-tocopherol, etc., are non-enzymatic
members.
Superoxide dismutase, a metalloenzyme is a most
proficient intracellular enzymatic antioxidant and is ubiquitously found in all
the aerobic organisms and in all the sub-cellular compartments susceptible to
ROS facilitated oxidative damage. Several environmental stresses often lead to
the increased generation of ROS, where SOD has been proposed to be a key enzyme
in plant stress tolerance and provide the first line of defense against the ill
effects of elevated levels of ROS. The SOD remove superoxide radical (a ROS
type) by catalyzing its dismutation via metal catalyzed Haber-Weiss
reaction (Gill and Tuteja 2010). Catalase, a tetrameric heme containing enzyme
is crucial for ROS clearing during the stressed circumstances (Gill and Tuteja
2010). It is a vital part of defense mechanisms that occurs predominantly in
the peroxisomes and glyoxysomes, where it dismutates hydrogen peroxides (H2O2),
another type of ROS. This enzyme is present in
all the aerobic organisms, and is essential for the abstraction of ROS produced
in peroxisomes by oxidases involved in β-oxidation of polyunsaturated fatty
acids, photorespiration and purine catabolism. It is also accountable for
removal of toxic peroxides (Chandrakar et al. 2019). Ascorbate peroxidase,
a multigene family enzyme is believed to play the ultimate protagonist in
scavenging of ROS, and protecting cells in higher plants. It can be cellular
compartment specific such as stromal APX (sAPX) and thylakoid-bound APX (tAPX)
in the chloroplasts, while in the glyoxysomes and peroxisomes exists as membrane-bound
APX (mAPX) and cytosolic APX (cAPX). As an essential element of ascorbate
glutathione cycle, APX catalyzes conversion of H2O2 into water
(Gill and Tuteja 2010; Hasanuzzaman et al. 2012). Guaiacol peroxidase, another
antioxidative enzyme, crumbles indole-3-acetic acid and is a part in the
biosynthesis of lignin and, defence against various biotic and abiotic stresses
by consuming H2O2. Guaiacol peroxidase prefers aromatic
electron donors preferably guaiacol and pyragallol, and oxidizing ascorbate at
the rate of about 1% that of guaiacol. The activity of it varies considerably
depending upon plant species and stress condition (Gill and Tuteja 2010).
To boost up the antioxidant
machinery, exogenous applications of melatonin (Mel) have been known to alleviate
heavy metal (HM) stress reactions in plants (Xalxo and Keshavkant 2019; Moustafa-Farag
et al. 2020). Melatonin, is soluble in both water and fat, and hence can spontaneously
penetrate the cell membranes and allocate themselves to any aqueous part preferably
the cytosol, nucleus and mitochondria. Analogous to auxin, it also regulates
growth of roots and shoots, wall acidification, cell division and elongation,
mitotic spindle formation, senescence, and so on (Hasan et al. 2015; Bałabustaet
al. 2016). It scavenges ROS directly and/or by enhancing activities of
antioxidants consequently stabilizing nucleic acids, proteins and lipids (Turk et
al. 2014; Hasan et al. 2015). Its metabolites own free radical scavenging
abilities, and are quite effective even at low concentrations (Yaduet al. 2018).
Therefore, valuation of its defending roles in contradiction of As stress is a
matter of scientific interest (Farouk and Al-Amri 2019; Siddiqui et al. 2020).
The conducted study
was aimed to assess As-stress alleviation ability of Mel on a significant crop Cicer arietinum L. by monitoring germination percentage, radicle length, biomass,
protein content and antioxidant enzymes.
Materials
and Methods
Seed Collection
and Treatments
For this study, the healthy non-infected and
uniform sized seeds of Cicer arietinum L.
were procured from local market of Raipur, Chhattisgarh, with the geographical
specifications 22°33ˈN and
21°14ˈN latitude, 82°6ˈE
and 81°38ˈ E longitude, and
298.15 meters above sea level. Randomly selected seeds of Cicer arietinum L. were surface sterilized with 1% (v/v) sodium
hypochlorite, for 15 min. The seeds were then thoroughly washed (5 times) with
MilliQ water (MW) (Millipore Gradient A-10, USA) and, fifty number of seeds
were allowed to germinate over double layered filter paper towels moistened
with sodium arsenite (source of As; 100 µM), Mel (50 µM) and As (100 µM) + Mel
(50 µM), in plastic boxes of 26 x 16 x 5 cm size, and kept in dark at 30-32ºC
(Chandrakar et al. 2017).
Germination
and Growth Assessments
After placing the seeds over filter paper
towels moistened with different treatment solutions, they were allowed to grow,
and germination was recorded on fifth day of treatments (Chandrakar et al. 2016). Germination tests were
performed with ten seeds each, and in three replicates. Germination data was
expressed as G%. On fifth day of various treatments, the radicles were
carefully removed from the germinating seeds of Cicer arietinum L. by cutting them with the help of a sharp razor
and were gently soaked on a blotting paper to make their surface dry. The
lengths of the growing radicles were measured on a graph paper.
To assess the biomass
content, ten radicles from each of the treatments were randomly harvested on
fifth day of their incubation. The FM (fresh mass) of the radicles was taken
using an electronic balance. For, dry mass of these radicles, they were kept in
a hot-air-oven at 60ºC for 72 h (Keshavkant et al. 2012). Each observation,
plotted in the figure is the mean ± SD of three individual replicates.
Extraction
of Protein and Enzymes
To extract protein
and enzymes, weighed amounts (0.2 g) of the radicles of Cicer arietinum L. were
extracted with 2 ml of 10 mM sodium phosphate buffer (pH 7.2), containing 1 mM
EDTA, 2 mM dithiothreitol and 0.2% (v/v) Triton X-100, and centrifuged at
14,000 rpm for 20 min at 4ºC. Clear supernatant was used for estimations of
protein and various enzymes (Chandrakar et al. 2017).
Estimation of Protein
Content of total
protein was estimated following the method of Bradford (1976). To the 20 µl of
extracted protein, equal amount of 20% (w/v) trichloroacetic acid was added,
and incubated for 30 min at laboratory conditions (25±2oC
temperature, 50±5% relative humidity). It was then centrifuged at 12,000 rpm
for 15 min at 4ºC. The pellet thus obtained was dissolved in 50 µl of 1 N NaOH.
An aliquot (20 µl) of it was mixed with 80 µl of MW and then 2 ml of Bradford
dye was added to it, and incubated in dark for 15 min. The absorbance of the
mixture was recorded at 595 nm by using UV-Vis spectrophotometer (Lambda-25,
Perkin Elmer, USA). Bovine serum albumin of 1 mg ml-1 concentration
was used as standard. Content of protein was expressed as mg g-1 FM.
Superoxide
Dismutase
Activity of SOD (EC 1.15.1.1) was determined by
taking 2.74 ml of Tris-HCl buffer (50 mM, pH 8.2) comprising 1 mM each of
diethylenetriaminepentaacetic acid and EDTA in a test tube, to which 0.2 ml of
enzyme extract was added. The reaction gets started by adding 60 μl of
pyrogallol (0.2 mM dissolved in 10 mM HCl), and the change in absorbance was
recorded at 420 nm after 6 min of incubation at laboratory temperature
(Marklund and Marklund 1974). The activity of the enzyme was expressed as Units
of SOD min-1 mg-1 protein.
Catalase
Activity of CAT (EC 1.11.1.6) was estimated
with 2.74 ml of potassium phosphate buffer (37.5 mM, pH 6.8) mixed with 60 μl
of enzyme extract. Enzyme activity was triggered by adding 200 μl of H2O2
(60 mM) and change in absorbance was recorded at 240 nm for 5 min at an
interval of 15 sec (Chance and Maehly1955). Catalase activity was calculated
using extinction coefficient 0.039 M-1 cm-1 and expressed
as nmol min-1 mg-1 protein.
Ascorbate Peroxidase
Activity of APX (EC 1.11.1.11) was assayed by
mixing 2.3 ml of potassium phosphate buffer (0.025 M, pH 7.0), 500 μl of
ascorbic acid (0.0025 M), 190 μl of EDTA (0.001 M) and 10 μl of enzyme extract.
Immediately after the addition of 10 μl of H2O2 (0.1 M)
to the assay mixture, initial absorbance was measured at 290 nm, and 20 min
after incubation final absorbance was recorded (Nakano and Asada 1981).
Activity was calculated using extinction coefficient 0.0028 M-1 cm-1
and data was expressed as mmol min-1 mg-1 protein.
Guaiacol Peroxidase
Activity of POD (EC 1.11.1.7) was measured taking
2.3 ml of sodium phosphate buffer (0.02 M, pH 6.4), 0.5 ml guaiacol (12 mM) and
0.2 ml enzyme extract. Reaction was triggered by adding 20 μl of 3% (v/v) H2O2
(Chance and Maehly1955). The change in absorbance was read at 470 nm after 3
min of incubation at laboratory temperature, and activity was expressed in
terms of µmol min-1 mg-1 protein.
Statistical
Analyses
All the experiments were performed twice with
three separate replications for the confirmation of results. The data obtained
were put to one-way ANOVA, and the mean differences were compared by Duncun’s
multiple range tests using SPSS software (Ver. 16.0).
Results
Germination
Percentage
Seeds of Cicer
arietinum L. exposed to As (100 µM), exhibited decrease in G% by 47.9% on
fifth day of sample harvest. However, application of Mel (50 µM) to seeds
increased the G% by 4.16%, as compared to control. On the other hand, application of Mel (50 µM)
along with As (100 µM) increased this trait by 26.66% as compared to As (100
µM) alone (Fig. 1).
Radicle Length
Lengths
of Cicer arietinum L. radicles
significantly decreased in response to As (100 µM) treatment. In contrary,
addition of Mel (50 µM) to seeds of Cicer
arietinum L. increased the radicle length by 40.81%, as compared to
control. However, supplementation of Mel (50 µM) along with 100 µM As mitigated
the As toxicity and exhibited the increase in radicle length by 165.94% on
fifth day, as compared to As (100 µM) alone (Fig. 2).
Biomass Accumulation
In line with the radicle
length, phytotoxic amount of As (100 µM) imposed deleterious impact on the
biomass of Cicer arietinum L. radicles, which was
assessed in terms of both FM and DM accumulations. On fifth day of analysis, accumulation of biomass
decreased (FM: 95.74% and DM: 83.33%) with 100 µM As addition, compared to
controls. In contrary, application of Mel (50 µM) to the Cicer arietinum L. increased the FM and DM by 32.78% and 22.16%
respectively, as compared to control. However, upon combined application of Mel
(50 µM) and As (100 µM), biomass was found to be increased by (FM: 261.9% and
DM: 233%) on fifth day of sample harvest, as compared to As (100 µM) alone
treatment (Figs. 3 and 4).
Protein Content
Exposure of Cicer
arietinum L. to As (100 µM) solution manifested massive loss in protein
turnover, as compared to control, on fifth day of sampling. On fifth day of
harvest, protein content was decreased by 43.5% in response to 100 µM As,
compared to control. In contrast, addition of Mel (50 µM) increased the protein
content by 23.65% as compared to control. However, co-application of As (100
µM) and Mel (50 µM) revealed increase in protein content by 39.74% as compared
to As (100 µM) alone (Fig. 5).
Superoxide Dismutase
Remarkable
fall in the activity of SOD was discernible on fifth day of sample harvest,
which was recorded to be 48% in case of 100 µM As, while was increased by 23%
in 50 µM Mel treated radicles, in comparison to control. On the other hand,
exogenous addition of Mel (50 µM) into As (100 µM) solution revealed 62.35%
increase in the SOD activity, compared to As (100 µM) alone treated tissues
(Fig. 6).
Catalase
Application
of As (100 µM) on Cicer arietinum L. radicles exhibited significant fall
(40%) in the activity of CAT, as compared to control, on fifth day of sampling.
However, exogenous addition of Mel (50 µM) alone, and in combination with As
(100 µM) solution stimulated its activity by 30% and 73% respectively, in
comparison to control and As (100 µM) alone respectively, on fifth day of
investigation (Fig. 7).
Ascorbate Peroxidase
Considerable
reduction in the APX activity was documented upon As (100 µM) treatment. On
fifth day of enzyme estimation, significant fall in the activity was
discernible, and noticed a drop of 43% in 100 µM As, in respect to control.
However, exogenous addition of Mel (50 µM) alone and along with As (100 µM),
increased the APX activity by 56% and 99.35% respectively, in comparison to
control and As (100 µM) alone, on fifth day of sampling (Fig. 8).
Guaiacol Peroxidase
Accumulated
data exemplified that the radicles of Cicer
arietinum L. exposed to As (100 µM) solution exhibited decreased activity
(77.7%) of POD on fifth day of sample harvest, compared to control. Whereas,
application of Mel (50 µM) increased the POD activity by 56.38%, compared to
the control. However addition of Mel (50 µM) along with As (100 µM), increased
POD activity by 161.2% in comparison to As (100 µM) alone, on fifth day of
sampling (Fig. 9).
Discussion
Growth
and development are vital processes to continue the survival and proliferation
of any vegetation. Presence of As more than that of threshold limit
in the soil or in irrigation water hinders the normal metabolism of plants, as
a result suppression in the seed germination and then radicle elongation,
lessening in number of leaves and leaf area thus decreased rate of
photosynthesis and biomass accrual, limited absorption of minerals, reduced
tissue viability and root elongation, stunted growth and poor productivity, etc.
(Chandrakar et al. 2017; Yadu et al. 2019). In the present work,
exposure of As revealed decreased germination percentage, radicle length and
biomass (both FM and DM) accrual in Cicer
arietinum L. radicles. In the current study, growth inhibitory impact of As
on germinating Cicer arietinum L. seeds
was analyzed.
It is evidenced from the current work that As (100 µM) significantly reduced the germination
percentage (47.9%),
radicle length (95%), FM (95.74%) and DM (83.33%) of Cicer
arietinum L. radicles as compared
with the MW-grown controls, on fifth day of treatments. Arsenic-induced
hampering in radicle length may be due to the disturbances in the rate of cell
division, cell elongation, and alterations in overall metabolic responses (Chandrakar
et al. 2017). Reduced rate of germination might also be due to the low water
uptake and scarcity of energy available, as As was shown to cause perturbations
in the cellular energy flow by replacing phosphate of the ATP molecule (Meharg
and Hartley-Whitaker 2002). Furthermore, declined biomass due to As was
possibly be due to increased permeability of the cell membranes,
consequentially enhanced leakage of cellular constituents/ basic nutrients
essentially required for energy generation, and optimum growth and development.
Arsenic-induced growth inhibition has also been observed in Glycine max L. and Cajanus cajan L. (Chandrakar et al. 2017; Yadu et al. 2019).
However, exogenous addition of Mel (50
µM),) into As (100
µM), markedly improved the growth traits (germination percentage: 26.66%, radicle
length: 165.94%, FM: 261.9% and DM: 233%) of five days old Cicer arietinum L. radicles, than that of As (100 µM) alone subjected samples. In recent past,
Mel was widely shown to improve
the seed germination responses and root/ shoot growth in different plants under
varied abiotic stresses (Yadu et al. 2018; Xalxo and Keshavkant 2019).
Arsenic-prompted
oxidative injury leads to oxidation of proteins and was estimated by measuring
total protein content. The results revealed that upon application of As (100
µM), there was a fall in protein content, compared to the control. Reduced
protein content was observed in As-exposed plant species by Chandrakar and Keshavkant
(2019). Abiotic stress has generally been shown to lead inhibition in the
syntheses of some of the proteins and promote others with a general trend of
decline in the overall content of protein. The decrease in protein content may
also be caused by binding of As to sulfhydryl groups of it and increased
protein degradation processes, as a result of enhanced protease activity under
stressful conditions (Chandrakar et al. 2017). On the other hand, the damaging
impacts of As in Cicer arietinum L. radicles
were attenuated substantially when Mel was added exogenously, hence; protein
content was increased substantially. As Mel was reported to enhance/ stabilize
the important amino acids during stressed condition that stabilizes protein
against oxidative damage (Siddiqui et al. 2020).
Alleviation of
As-induced oxidative damage via
stimulation of antioxidant enzymes is a significant approach of plants to
overcome from As-toxicity (Chandrakar et al. 2020). Activities of SOD, CAT, POD
and APX were significantly declined in response to As-exposure and could be one
of the possible reasons for enhanced oxidative injury (Yadu et al. 2018).
Reduction in the activities of these enzymes indicated that they were
insufficient to completely detoxify the ROS produced in response to As-stress (Chandrakar
et al. 2017). This might be due to the alteration in the assembly of enzyme
subunits and/or ineffective enzyme synthesis (Hasanuzzaman and Fujita 2013). Alteration
in the activities of these enzymes has also been reported by Chun-xi et al.
(2007) and Chandrakar et al. (2016) in As-exposed seeds/ seedlings of Triticum aestivum L. and Glycine max L. respectively. However,
exogenous application of Mel increased the activities of tested enzymes
considerably which resulted in decreased oxidative damage in As-subjected Cicer arietinum L. radicles. This compound is well known to
stimulate/ enhance the activities of antioxidant enzymes in abiotic stressed
plants and their parts (Yadu et al. 2018; Xalxo and Keshavkant 2019).
Conclusions
In the current piece of research, toxicity of As in five days old
radicles of Cicer arietinum L. was monitored which revealed significant reductions in germination
percentage, length of radicle, as well as biomass (FM and DM) accumulation. Our
findings clearly support the hypothesis that there is an imbalance between ROS
generation and its scavenging enzymes under As-stress. This imbalance resulted
due to the existence of “oxidative stress situation” in growing Cicer arietinum L. radicles. In response to As-induced oxidative burst, the activities of
antioxidant enzymes; SOD, CAT, POD and APX were found to be declined under
As-toxicity. On the other hand, exogenous application of Mel, into growth
solutions, significantly compensated the As-imposed losses/ changes in growing Cicer arietinum L. radicles, therefore; revealed better germination percentage, radicle
length, biomass accumulation and increased antioxidant activities.
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