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Evaluating the imazethapyr herbicide
mediated regulation of phenol and
glutathione metabolism and antioxidant
activity in lentil seedlings
Rajeev Kumar
1
, V. Visha Kumari
2
, Ranjit Singh Gujjar
3
, Mala Kumari
4
,
Sanjay Kumar Goswami
5
, Jhuma Datta
6
, Srikumar Pal
7
,
Sudhir Kumar Jha
8
, Ashok Kumar
9
, Ashwini Dutt Pathak
3
,
Milan Skalicky
10
, Manzer H. Siddiqui
11
and Akbar Hossain
12
1Division of Plant Physiology & Biochemistry, Indian Institute of Sugarcane Research, Lucknow,
Uttar Pradesh, India
2Agronomy, Central Research Institute for Dryland Agriculture, Hyderabad, Telangana, India
3Crop Improvement, Indian Institute of Sugarcane Research, Lucknow, Uttar Pradesh, India
4Integral Institute of Agriculture Science and Technology, Integral University, Lucknow,
Uttar Pradesh, India
5Crop Protection, Indian Institute of Sugarcane Research, Lucknow, Uttar Pradash, India
6Department of Agricultural Biochemistry, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur,
West Bengal, India
7Agricultural Biochemistry, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, West Bengal,
India
8Division of Plant Biotechnology, Indian Institute of Pulses Research, Kanpur, Uttar Pradesh,
India
9Division of Biochemistry, Indian Agricultural Research Institute, New Delhi, India
10 Department of Botany and Plant Physiology, Faculty of Agrobiology, Food, and Natural
Resources, Czech University of Life Sciences Prague, Prague, Czechia
11 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh,
Saudi Arabia
12 Soil Science, Bangladesh Wheat and Maize Research Institute, Dinajpur, Bangladesh
ABSTRACT
The imidazolinone group of herbicides generally work for controlling weeds by
limiting the synthesis of the aceto-hydroxy-acid enzyme, which is linked to the
biosynthesis of branched-chain amino acids in plant cells. The herbicide imazethapyr
is from the class and the active ingredient of this herbicide is the same as other
herbicides Contour, Hammer, Overtop, Passport, Pivot, Pursuit, Pursuit Plus, and
Resolve. It is commonly used for controlling weeds in soybeans, alfalfa hay, corn, rice,
peanuts, etc. Generally, the herbicide imazethapyr is safe and non-toxic for target
crops and environmentally friendly when it is used at low concentration levels. Even
though crops are extremely susceptible to herbicide treatment at the seedling stage,
there have been no observations of its higher dose on lentils (Lens culinaris Medik.) at
that stage. The current study reports the consequence of imazethapyr treatment on
phenolic acid and flavonoid contents along with the antioxidant activity of the
phenolic extract. Imazethapyr treatment significantly increased the activities of
several antioxidant enzymes, including phenylalanine ammonia lyase (PAL), phenol
oxidase (POD), glutathione reductase (GR), and glutathione-s-transferase (GST), in
lentil seedlings at doses of 0 RFD, 0.5 RFD, 1 RFD, 1.25 RFD, 1.5 RFD, and 2 RFD.
Application of imazethapyr resulted in the 3.2 to 26.31 and 4.57–27.85% increase in
How to cite this article Kumar R, Kumari VV, Gujjar RS, Kumari M, Goswami SK, Datta J, Pal S, Jha SK, Kumar A, Pathak AD, Skalicky
M, Siddiqui MH, Hossain A. 2024. Evaluating the imazethapyr herbicide mediated regulation of phenol and glutathione metabolism and
antioxidant activity in lentil seedlings. PeerJ 12:e16370 DOI 10.7717/peerj.16370
Submitted 29 June 2023
Accepted 8 October 2023
Published 3 January 2024
Corresponding authors
V. Visha Kumari,
visha.venugopal@gmail.com
Akbar Hossain,
akbarhossainwrc@gmail.com
Academic editor
Ahmet Tansel Serim
Additional Information and
Declarations can be found on
page 17
DOI 10.7717/peerj.16370
Copyright
2024 Kumar et al.
Distributed under
Creative Commons CC-BY 4.0
mean phenolic acid and flavonoid content, respectively, over control. However, the
consequent fold increase in mean antioxidant activity under 2, 2-
diphenylpicrylhdrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assay
system was in the range of 1.17–1.85 and 1.47–2.03%. Mean PAL and POD activities
increased by 1.63 to 3.66 and 1.71 to 3.35-fold, respectively, in agreement with the
rise in phenolic compounds, indicating that these enzyme’s activities were modulated
in response to herbicide treatment. Following herbicide treatments, the mean thiol
content also increased significantly in corroboration with the enhancement in GR
activity in a dose-dependent approach. A similar increase in GST activity was also
observed with increasing herbicide dose.
Subjects Agricultural Science, Plant Science
Keywords Herbicide, Imazethapyr, Lentil, Antioxidants, Seedlings, Phenolic acid
INTRODUCTION
Lentil (Lens culinaris Medik.), is an edible cool-season legume, which contains ample
amounts of high-quality protein. It also possesses carbohydrates, micronutrients, vitamins,
phenolics, and flavonoids (Khazaei et al., 2019). Due to an immense weed invasion during
the early crop growth cycle, the growth and development of the lentil plant is severely
impeded which lowers yield and its contributing qualities (Jaswal & Menon, 2020). Based
on the environmental factors, weed diversity, and density, the losses incurred are in the
range of 20–80% (Balech et al., 2022). Proper management practices such as sowing
method and time, cover crops, crop rotations, and varietal selection are customarily used
to retard the growth and biomass of the weed and improve the lentil yield. However, these
approaches are insufficient to curb weed interference effectively (Pala, Mennan & Demir,
2018). Compared to other methods, the effectiveness of herbicide treatment as of yet
appears to be very high. It also acts quickly and requires little financial investment to
control weeds (Singh & Singh, 2017). Imazethapyr (IM) application to manage the broad
spectrum of weed flora has been advised as a means of reducing and controlling the weed
threat in pulse crops (Duary, Dash & Teja, 2016). Imazethapyr belongs to the
imidazolinone family and is applied as a post-emergence herbicide; a characteristic feature
of this class includes its effectiveness at a lower dose, larger selectivity to various crops and
lesser mammalian toxicity (Tranel & Wright, 2002). The selectivity of the imazethapyr is
unique to control the multiple weed class at a lower rate of application (Presotto et al.,
2012). Usually, imazethapyr application is found to be harmless to target crops and
ecofriendly, when applied in lower amounts (Hoseiny-Rad & Aivazi, 2020).
Imazethapyr-mediated consequences on plant growth are primarily exerted through
inhibition of the rate-limiting enzyme involved in branched-chain amino acids (BCAAs)
synthesis (valine, leucine, and isoleucine) i.e., acetolactate synthase (ALS) (Qian et al.,
2015). Secondary consequences such as disruption in protein synthesis and cell division are
associated with Imazethapyr-induced ALS inhibition. In addition to these issues,
imazethapyr builds up in the plant’s meristematic areas after being applied topically, which
slows down the growth and development of the plant (Hoseiny-Rad & Aivazi, 2020). Even
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 2/22
though, ALS inhibition affects nitrogen metabolism through the reduction in protein
synthesis rate as a consequence of a transitory halt in BCAAs synthesis (Zabalza et al.,
2006). The plant’s sessile nature restricts it from facing unavoidable environmental
stresses, like drought, heat, freezing, high salt, xenobiotics contaminants including
herbicide, etc., which are the major abiotic factors that impact the productivity of the crop
(Kumar et al., 2023). This results in substantial economic loss further raises the nutritional
security issue (Nianiou-Obeidat et al., 2017). To survive under such stressful conditions
plants have developed a potential defense mechanism that combats these stress
eventualities (Proietti et al., 2019). The adaptive response mechanism of plants against
several abiotic stresses implicates a complex system that is regulated through the
coordinated action of multiple signalling compounds. These compounds include reactive
carbonyl species (RCS), reactive oxygen species (ROS), phytohormones and reactive
nitrogen species (Fancy, Bahlmann & Loake, 2017). ROS generated in plants during their
exposure to herbicides creates a stress condition for plants (Hassan & NematAlla, 2005;
NematAlla, Hassan & El-Bastawisy, 2008). Antioxidants are a crucial component of the
plant defence mechanism that protects plants from oxidative stress brought on by
herbicidal stress. The coordinated action of both enzymatic and non-enzymatic
antioxidants in the plant system, produced upon exposure, controls ROS elimination
(Grewal et al., 2022). Phenol and glutathione and their associated metabolism are excellent
mechanisms to turn down the stress impact caused by the herbicide and impart tolerance
against them. Regulation of phenol metabolism is primarily monitored through the
phenylalanine ammonia-lyase (PAL) action and activity, the key enzyme involved at the
entry valve of phenol metabolism (Kong, 2015). Furthermore, these synthesized phenol
molecules oxidized through the coordinated action of oxidative enzymes such as
polyphenol oxidase (PPO) and peroxidase (POD) (Singh et al., 2022). Besides, glutathione
metabolism-associated enzymes viz. glutathione-s-transferase (GST) and glutathione
reductase (GR) are also important players in the detoxification of ROS generated through
herbicidal-induced oxidative stress (Tseng, Ou & Wang, 2013). Identification of herbicide
dose and its associated tolerance mechanism at the early phase of the crop cycle in lentil
would have potential use to recommend in lentil cultivation practice for control of weeds
without compromising the plant growth and yield. Taking all these into consideration this
present study was conducted to assess the dose-dependent response of imazethapyr on
phenol and glutathione metabolism and its associated antioxidant potential in lentil
seedlings at different sampling hours after its application. Furthermore, the current study
will decipher the phenols and glutathione-associated tolerance mechanism against
imazethapyr in lentils at the seedling stage.
MATERIALS AND METHODS
Experimental setup/establishment of settlings
The present investigation was carried out in a pot culture-based experiment under
controlled conditions (net house) at the division of Agricultural Biochemistry, Bidhan
Chandra Krish Vishwavidalaya, Mohanpur, India. Before sowing, seeds of lentil (cv.
Moitree (WBL 77)) were sterilized (surface sterilization) with 3% sodium hypochloride up
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 3/22
to 10 min, following the thorough washing using distilled water. Properly washed seed
subjected to soaking (8 h), and transferred in the pots of 30 cm width × 30 cm top height.
Following the transfer of soaked seeds, seedlings were uniformly thinned to 30 plants in
each plot after 10 days. Further, pots were divided and allocated to six groups, control (1),
while the rest of the pots (5) were used to implicate the five different imazethapyr (10 SL)
doses as outlined below. T0: Untreated control; T1: imazethapyr @ 12.5 kg ai/ha (0.5 RFD,
0.5 multiples of Recommended Field Dose); T2: imazethapyr @ 25.0 kg ai/ha (RFD,
Recommended Field Dose); T3: imazethapyr @ 31.25 kg ai/ha (1.25 RFD, 1.25 multiples of
the Recommended Field Dose); T4: imazethapyr @ 37.50 kg ai/ha (1.50 RFD, 1.50
multiples of the Recommended Field Dose); T5: imazethapyr @ 50.00 kg ai/ha (2.0 RFD,
2.0 multiples of Recommended Field Dose). Depending on the surface area of the pot and
amount of soil on a per hectare basis, the required amount of imazethapyr was calculated
for each herbicide dose, and each herbicide dose (amount) was then solubilized with the
adequate quantity of water before being sprayed using a mechanical sprayer (knapsack) in
a cross-wise direction. Each treatment was replicated in quadruplicate in a completely
randomized block design. Collection of lentils shoot just before the application of
imazethapyr (0 h) and subsequently at regular intervals of 30 h up to 120 h after treatment
(HAT) was performed with proper care. The collected shoots were rinsed with a copious
amount of water to avoid the soil particles and dried using tissue paper.
Analysis of phenylalanine ammonia-lyase
The enzyme extract was prepared by grinding 1 g of fresh tissue (whole seedlings) in 0.1 M
phosphate buffer (10 ml pH 7.5). Additionally, 2% polyvinylpyrrolidone and triton-x
(0.25%) were used to prepare the extraction buffer. The extraction of plant samples
(seedlings) was performed in pestle and mortar (pre-chilled). The homogenate obtained
through maceration was centrifuged at 10,000 rpm for 30 min at 4 C, and the obtained
supernatant was used to conduct the enzyme assay. The extracted enzyme source was kept
in an ice bath, before the enzyme assay set-up. Estimation of PAL activity was performed
by mixing 0.1 M Tris-HCl pH 8.8 (1.9 ml), 1 ml substrate (0.01 M phenylalanine) and
freshly prepared enzyme extract (chilled) (0.1 ml). Monitoring of change in the absorbance
(ΔA) performed at a regular interval of 5 min up to 30 min at 270 nm. Further, estimation
of the activity was carried out using the method of Burrell & Rees (1974) with required
modifications. The specific activity of the enzyme was calculated by using the standard
curve (trans-cinnamic acid). The specific activity of PAL against herbicide (imazethapyr
dose) was expressed in µmol trans-cinnamic acid produced h
−1
mg
−1
of protein.
Analysis of phenol oxidase
Estimation of phenol oxidase was performed using the method of Shannon (1966) with
slight modifications. The reaction mixture for enzyme assay contains potassium phosphate
buffer pH 7.5 (2.65 ml), methanol dissolved guaiacol (4%) (0.15 ml), H
2
O
2
(1%) (0.15 ml)
and 0.05 ml enzyme source. Furthermore, mixing of the reaction constituent was done
(bottom-top shaking approach) in a fraction of a second. The observation of absorbance
change (ΔA) was monitored at a regular interval of 30 s to 3 min at 470 nm. Calculation of
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 4/22
phenol oxidase was accomplished by using the absorption coefficient of the tetra guaiacol
at 470 nm (26.6 mM
−1
cm
−1
). The specific peroxidase activity expressed in µmol of guaiacol
oxidized min
−1
mg
−1
of protein.
Analysis of total phenol and flavonoid contents
The total phenolic acid content of lentil shoots was estimated by using Folin-Ciocalteau
Reagent (FCR) following the method of Vinson (Vinson et al., 1998) with required
modification. Briefly, the dried powdered (0.1 g) lentil shoot sample was mixed in 15 ml
1.2 N HCl prepared in aqueous methanol (50%). Furthermore, the methanolic extract was
subjected to heat (2 h at 90 C). Extracted material following the cooling (kept at room
temperature) was subsequently centrifuged for 30 min at 10,000 rpm. Furthermore, the
supernatant in the volumetric flask and let it evaporate to become dry, finally, the volume
was made to 25 ml with double distilled water. For the evaluation of phenol content, a
particular aliquot volume was taken, further dilution (3 ml) with double distilled water was
carried out in a test tube, and finally, the addition of 0.5 ml FCR in the diluted methanolic
extract was accomplished. Subsequently, sodium carbonate (10%, 2 ml) was added after
5 min of FCR addition. Then, it was placed in a water bath for 7 min at 65–70 C. The
reaction was stand allowed to cool (room temperature) and the absorbance of the solution
was taken at 650 nm. A standard curve using 8 different phenolic acids, namely gallic acid,
chlorogenic acid, hydroxybenzoic acid, p-protocatechuic acid, vanillic acid, caffeic acid,
p-coumaric acid, and ferulic acid was prepared as per the method ascribed by Alla &
Younis (1995) with slight modification. Each of the phenolic acids represents its respective
concentration (5 ppm), which corresponds to the final concentration of total phenolic
acids at 40 ppm. The standard curve was used for the calculation of total phenolic acid in
lentil shoots (seedlings) and expressed in mg phenolic acid g
−1
.
In addition, a standard of flavonoids was prepared following the Folin-Ciocalteau assay
using the mixture of seven different flavonoids comprising apigenin, myricetin, quercetin,
genistein, catechin, kaempferol, and diadzein. All but two of the flavonoids were at a
concentration of 5 ppm each, while apigenin and myricetin were added at a concentration
of 2.5 ppm each, which corresponds to a total flavonoid concentration of 30 ppm. Thus,
the flavonoid content of lentil shoots was measured based on the standard curve of
flavonoids and expressed as mg flavonoid g
−1
sample.
Measurement of antioxidant activity
Because of the difficulty in the measurement of phenolic composition with their
antioxidant role in plant tissue, antioxidant activity may conveniently be signified as a
measure of the quality of phenol. The antioxidant action of a substance is a measure of the
capability to transfer an electron either in the form of hydrogen atom transfer (HAT) or a
single electron transfer (SET). In the current study, total phenolic extracts were used to
determine the antioxidant activity of lentil seedlings by using DPPH and FRAP assay,
which uses antioxidant mechanisms involving HAT and SET, respectively.
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 5/22
DPPH assay
DPPH is a neutral radical, which is extensively used to measure antioxidant activity in
clinical studies. Hydrogen atom transfer from the substrate reduces the DPPH radical,
which is accompanied by a decrease in the intensity of colour as well as absorbance of the
solution. The DPPH assay was executed using the method adopted by Braca et al. (2001).
The reaction mixture consisting of 150 µL of the aliquot of total phenol extract and
2,850 µL of the DPPH solution (0.004%) was taken and properly mixed by hand-shaking.
Furthermore, it was kept at normal temperature for 30 min in a dark place. The absorbance
of the solution was taken at 517 nm, for blank distilled water was used along with DPPH.
The standard curve was prepared by using 150 µL of each of the different concentrations of
Trolox and 2,850 µL of 0.004% DPPH solution. The antioxidant activity, also termed as
DPPH-generated radical scavenging capacity is expressed as a milligram of trolox
equivalent per gram of fresh weight (mg TE g
−1
FW).
FRAP assay
FRAP system of the antioxidant assay has relied on the capability of phenol extract to
reduce Fe (III), which was measured according to the Benzie and Strain (Benzie & Strain,
1996) method with slight modification. Changes in the absorbance owing to the
blue-coloured compound Fe(II)-tripyridyltriazine formation from the colourless parent
compound containing the oxidized form of Fe (III), were assayed in the FRAP system, and
the presence of the unknown concentration in phenolic extract was monitored.
Preparation of the FRAP reagent was accomplished through the mixing of acetate buffer
(0.1 M, pH 3.6), 2,4, 6-tri (2-pyridyl)-s-triazine (TPTZ) (10 mM) and ferric chloride
(20 mM) in 10:1:1 (v/v/v) proportions. The reaction mixture comprising 2,850 µL reagent
(FRAP) and 150 µL sample aliquot was kept at normal temperature (30 min). Thereafter,
absorbance was taken at 593 nm. Different concentration of Trolox was used for the
standard curve preparation. Finally, results were presented as milligrams of trolox
equivalent to per gram of fresh weight (mg TE g
−1
FW).
Analysis of enzyme activity of glutathione-s-transferase and
glutathione reductase
To assay the enzyme activity of both GST and GR, the enzymatic extract was prepared by
macerating the fresh tissue (1 g whole seedlings) in 10 ml of phosphate buffer (0.1 M pH
7.5). Additionally, 7.5% PVP, 14 mM β-mercaptoethanol and 2 mM EDTA were added to
the extraction buffer, and crushing of plant tissue (whole seedlings) was performed in a
pre-chilled mortar pestle. The homogenate obtained was transferred into the centrifuge
tube. Subsequent to extraction plant samples (seedlings) were subjected to centrifuge for 30
min at 10,000 rpm and 4 C. The obtained supernatant was used to assay both GST and GR
activities. The activity of glutathione reductase was assayed following the procedure of Rao,
Paliyath & Ormrod (1996) with minor modification. The principle of this method relies
upon the oxidation of assimilatory power (NADPH) through oxidized glutathione (GSSG).
The reaction set up for the GR assay comprises Tris-HCl buffer (2.3 ml, pH 9.0), 0.1 ml
GSSG (5.44 mM), 0.1 ml EDTA and 0.2 ml enzyme source. The reaction was commenced
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 6/22
by adding 0.2 ml of NADPH (0.2 mM). Subsequently, the absorbance of the solution was
observed at 340 nm at a regular interval (1 min up to 5 min). Furthermore, the activity of
GR was calculated by using the NADPH extinction coefficient (6.2 mM
−1
cm
−1
) at 340 nm.
The specific GR activity was presented in µmol of NADPH oxidized min
−1
mg
−1
of protein.
The reaction mixture in GST assay comprises 2.4 ml of potassium phosphate buffer (pH
6.5), 0.2 ml of GSH (5.0 mM), 0.2 ml of 1 mM CDNB and enzyme extract (0.2 ml).
Subsequently, measurement of absorbance (OD) was taken at 340 nm at the interval of
1 min and the last reading was taken at 5 min. The specific activity of GST was calculated
based on the CDNB extinction coefficient (9.6 mM
−1
cm
−1
) following the Ando methods
(Ando et al., 1988), with required modification which is based on the conjugation of CDNB
to GSH.
Analysis of total thiol
The amount of total thiol in lentil seedlings was measured using the method of
Maas et al. (1987) through minor modifications. The homogenization of the plant
sample (0.5 g) was performed in 0.02 M EDTA. Extraction was followed by centrifugation
of the homogenate at 10,000 rpm for 30 min at 4 C. The obtained supernatant was used
for the thiol estimation. To set up the reaction 0.5 ml of sample extract, 0.7 ml buffer
(Tris-HCl, 0.2 M and pH 7.5), 0.3 ml DTNB (10 mM) and cold methanol (3.5 ml).
Furthermore, it was incubated (15 min) at normal temperature and absorbance was
recorded at 412 nm. The calculation of total thiol concentration was done using the
standard curve of reduced glutathione prepared for different concentrations. The result
was expressed as µmole of GSH g
−1
fresh tissue (µmole GSH g
−1
FW). Statistical analysis
was performed by using the SPSS Professional Statistics ver. 7.5 (SPSS Inc., Irvine, CA,
USA). Calculation and graphical presentation of the data executed in M/S Excel software.
RESULTS
PAL and POD activity in lentil
Phenolic compound metabolism is chiefly regulated and controlled through the
coordinated action of various enzymes involved in the synthesis as well as the breakdown
of the compounds. Conversion of L-phenylalanine (L-Phe) to cinnamic acid (trans-CA) is
catalyzed through phenylalanine ammonia lyase (PAL, EC 4.3.1.24) via non-oxidative
deamination reactions. Peroxidase (POD) and polyphenol oxidase (PPO) also play a major
role in phenolic compound metabolism.
Therefore, PAL and POD activity was evaluated in the current investigation to acquire
more evidence on imazethapyr-induced modifications in phenol metabolism as a stress
reaction. Figures 1A and 1B, respectively, show the results for PAL and POD activity as
affected by the use of various imazethapyr treatments. The results show that mean PAL
and POD activity irrespective of sampling hours increased significantly above control in
response to increasing concentration of imazethapyr treatments. The increase in mean
PAL and POD activity showed a 1.63–3.66 and 1.71–3.35-fold variation as compared to the
control. Moreover, the mean PAL activity did not show any significant variation
between 0 HBT and 30 HAT, but thereafter increased significantly throughout the
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 7/22
experimental period. Regardless of treatments, PAL activity increased by 4.67-fold on 120
HAT as compared to 0 HBT. Contrary to PAL activity, significant differences in mean
POD activity were observable and increased progressively throughout the experimental
period. The enhancement in POD activity varied from 1.04–3.02 folds as compared to 0
HBT. Thus, both enzymatic activities are modulated by the treatment and sampling hours.
In addition, the interaction between treatment (imazethapyr doses) and sampling hours
was also significant. PAL, a marker of several kinds of abiotic stresses including herbicides,
channels aromatic amino acids, chiefly phenylalanine to diverse phenolic compounds with
equally diverse biological functions, which are related to ameliorating diverse
environmental challenges. In the current study, a substantial increase in the mean PAL
activity in response to an increased rate of the imazethapyr application and these effects are
pronounced with the progression of the growth stage are noteworthy.
Phenolic acid and flavonoid content in lentil
Following the application of imazethapyr at five different concentrations, phenolic acid
and flavonoids were analyzed periodically before and after herbicide treatment. The results
are summarized in Figs. 2A and 2B. The results obtained indicate that both mean phenolic
acid and flavonoid content in lentil shoots increased significantly with the increasing
application rate of imazethapyr. The mean phenolic acid over different sampling hours
varied from 0.152–0.192 mg g
−1
FW, while the corresponding values for flavonoids were
between 0.219 and 0.280 mg g
−1
FW. Hence, the application of imazethapyr resulted in the
elevation of phenolic acid to the extent, which ranged from 3.2% to 26.31% over control,
with maximum elevation recorded at 2 RFD and least at 0.5 RFD. A similar increase in
Figure 1 (A) Phenylalanine ammonia-lyase (PAL) and (B) phenol oxidase (POD) activity of imazethapyr treated seedlings at 0 HBT and 30,
60, 90 and 120 HAT under treatments of 0, 0.5, 1, 1.25, 1.5 and 2 RFD. HBT, hours before treatment; HAT, hours after treatment; RFD,
recommended field dose. The vertical error bar represents a standard error (n= 4). Full-size
DOI: 10.7717/peerj.16370/fig-1
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 8/22
flavonoid content, varying from 4.57–27.85%, was also observed depending on the
herbicide dose. However, on the other side, the mean phenolic acid and flavonoid content
over different treatments increased significantly with the progression of growth at early
stages. During 120 h, the phenolic acid and flavonoid content increased over 0 HBT by
38.41 and 37.62%. The interaction outcome among herbicide dose and sampling hours also
revealed significant involvement, indicating that phenolic acid and flavonoid content in
lentil shoots changes depending on application rate as well as growth stage.
Antioxidant activity in lentil
Diverse types of biomolecules known as antioxidants strongly resist the oxidant molecules
produced by free radicals and oxidation driven by them, protecting the cellular
biomolecules, even when present in minute quantities alongside other oxidizable
substrates. Various biomolecules of diverse structural groups are recognized to act as an
antioxidant, which includes phenols, tocopherols, ascorbic acid, glutathione, etc.
The shared central feature in these compounds lies in their ability to scavenge radical
species. In the present study, the total phenolic extracts were analyzed for antioxidant
activity using DPPH radical and FRAP, which exemplify a hydrogen atom transfer (HAT)
and Single Electron Transfer (SET) mechanism of antioxidant reaction respectively. These
mechanisms specify and exemplify the antioxidant reaction catalyzed by them.
The antioxidant activity in phenolic extract executed through DPPH and FRAP assay are
summarized in Figs. 3A and 3B respectively. It is evident from Figs. 3A and 3B, that mean
Figure 2 (A) Total phenolic acid (mg g
−1
) FW and (B) flavonoids content (mg g
−1
) FW of imazethapyr treated seedlings at 0 HBT and 30, 60,
90, and 120 HAT under treatments of 0, 0.5, 1, 1.25, 1.5 and 2.0 RFD. HBT, hours before treatment; HAT, hours after treatment; RFD,
recommended field dose. The vertical error bar represents a standard error (n= 4). Full-size
DOI: 10.7717/peerj.16370/fig-2
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 9/22
antioxidant activity under both assay systems showed significant differences across
different treatments and increased with increasing rates of application.
GR and GST activities in lentil
The effect of imazethapyr on GR and GST activities is summarized in Figs. 4A and 4B,
respectively. Both GR and GST activities enhanced substantially with the increased
application rate of herbicide and progression of growth. The mean GR activity varied from
27.995–66.041 µmol of NADPH oxidized min
−1
mg
−1
protein, which represented an
increase in GR activity ranging from 55.79 to 136.24% over the untreated control.
However, the corresponding increases in GST activity were in the range of 61.30% to 157%
over control. The mean GR activity showed a decline on 30 HAT and increased thereafter,
while the mean GST activity increased progressively throughout the experimental period.
The GSH/GSSG proportion was higher with the pretilachlor-treated plant while least in
metribuzin, GR and GST activities were stimulated more with pretilachlor than metribuzin
in maize leaves. These herbicides, thus, induced oxidative stress differentially in maize,
which is more, pronounced with metribuzin than with pretilachlor. Henceforth, it
indicates the differential tolerance resulting from the enhancement in GSH content and
stimulation of its associated enzyme activity. The elevation in the production of
antioxidants such as phenol, and thiols and the prominent increase in the antioxidant
Figure 3 Total phenol extract (mg TE g
−1
) FW in imazethapyr-treated seedlings using (A) DPPH and (B) FRAP assay at 0 HBT and 30, 60, 90,
and 120 HAT under treatments of 0, 0.5, 1, 1.25, 1.5 and 2.0 RFD. HBT, hours before treatment; HAT, hours after treatment; RFD, recommended
field dose. The vertical error bar represents a standard error (n= 4). Full-size
DOI: 10.7717/peerj.16370/fig-3
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 10/22
enzyme associated with these compounds like GR, GST, PAL and POD, appear to be the
general strategies of the plant defense system to restrict the toxic peroxidation in plants.
Thiol content in lentil
The results relating to total thiol content in lentil shoot following the application of
imazethapyr at five different doses is depicted in Fig. 5, which revealed that the mean thiol
content of lentil shoot showed significant differences depending on the treatment and
Figure 5 Total thiol content (µmol GSH/g FW) of imazethapyr-treated seedlings at 0 HBT, and 30,
60, 90, and 120 HAT under treatments of 0, 0.5, 1, 1.25, 1.5, and 2 RFD. HBT, hours before treatment;
HAT, hours after treatment; RFD, recommended field dose. The vertical error bar represents the standard
error. Full-size
DOI: 10.7717/peerj.16370/fig-5
Figure 4 (A) Glutathione reductase (GR) and (B) glutathione-s-transferase (GST) activity in imazethapyr treated seedlings at 0 HBT and 30,
60, 90, and 120 HAT under treatments of 0, 0.5, 1, 1.25, 1.5 and 2 RFD. HBT, hours before treatment; HAT, hours after treatment; RFD,
recommended field dose. The vertical error bar represents a standard error (n= 4). Full-size
DOI: 10.7717/peerj.16370/fig-4
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 11/22
sampling hour. The mean thiol content regardless of sampling hours was recorded higher
with 2 RFD (5.22 µmol GSH/g FW), and that decreased with decreasing rates of
application. The thiol content was recorded lowest with 0.5 RFD (3.94 µmol GSH/g FW).
DISCUSSION
The imidazolinone group of herbicides generally work for controlling weeds by limiting
the synthesis of the aceto-hydroxy-acid enzyme, which is linked to the biosynthesis of
branched-chain amino acids in plant cells (Presotto et al., 2012). The herbicide
imazethapyr is from the class and the active ingredient of this herbicide same as other
herbicides Contour, Hammer, Overtop, Passport, Pivot, Pursuit, Pursuit Plus, and Resolve.
It is commonly used for controlling weeds in soybeans, alfalfa hay, corn, rice, peanuts, etc.
Generally, the herbicide imazethapyr is safe and non-toxic for target crops and
environmentally friendly when it is used at low concentration levels (Qian et al., 2015;
Duary, Dash & Teja, 2016;Hoseiny-Rad & Aivazi, 2020). Although crops are extremely
susceptible to herbicide treatment at the seedling stage, there have been no observations of
its higher dose on lentil (Lens culinaris Medik.) at that stage (Hanson & Thill, 2001;Grewal
et al., 2022).
PAL and POD activity in lentil
Imazethapyr-induced dose-dependent, concurrent increase in the PAL and POD activity
in the present investigation has been observed. This concomitant enhancement in activity
indicates the possibility of a metabolic shift from primary to secondary to defend the plant
against imazethapyr-generated free radicals. This shift in the present study is visualized
through an increase in secondary metabolic compounds such as phenolic acid and
flavonoid contents. It is likely to be concluded that the buildup of phenolic acid and
flavonoid in lentil shoots with increasing herbicide dose cannot be explained solely based
on enhanced PAL activity. Moreover, increased POD activity illustrates the availability of
oxidizable substrates in the form of phenolic compounds upon increasing the herbicide
dose. These increased substrate availabilities for the POD manifested the limiting role of
PAL in phenol metabolism. However, enhanced POD activity may further lead to a more
diverse class of compounds that enrich the plant defence armoury to face the
herbicide-associated consequences. Therefore, PAL appears to be a key enzyme to regulate
phenol accumulation in lentil seedling and POD acted in tandem. The enhanced activity of
PAL further confirmed with increasing phenolic acid is reported in herbicide-treated
seedlings of soybean and maize (Alla & Younis, 1995). Thus, imazethapyr-induced
stimulation in the PAL activity complemented with increased phenol content in our study
represents additional evidence for the effect of herbicides on phenol metabolism.
Enhanced PAL and TAL (Tyrosine ammonia lyase) in response to imazethapyr in soybean
root and shoot has been reported as a stress symptom (Scarponi, Martinetti & Nemat Alla,
1996). A large number of herbicides, including acifluorfen (Kömives & Casida, 1982),
metolachlor (Scarponi, Alla & Martinetti, 1992), and alachlor (Molin, Anderson & Porter,
1986), also caused increased PAL activity, which is concerning for this relationship.
Therefore, PAL appears to be a key enzyme to regulate phenol accumulation in lentil
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 12/22
seedling and POD acted in tandem. which is supported by Scarponi’s report (Scarponi, Alla
& Martinetti, 1992). Metribuzin induced an increase in the activities of peroxidases such as
ascorbate peroxidase, guaiacol peroxidase, and the polyphenol oxidase observed in wheat
(Rajabi et al., 2012). Moreover, Islam et al. (2017), suggested the role of butachlor at a
lower dose in the enhancement of GPX activity in rice (cv. ZJ 88), while at the higher dose,
the activity of the enzyme was downregulated. Thus, our results do not align with the
observation of Islam et al., which perhaps describes the substantial heterogeneity among
the crop species and the difference in the nature of herbicides. Imazethapyr-induced
enhanced expression of PAL isoforms in Arabidopsis root in a proteomics-based study has
been observed (Qian et al., 2015). Biosynthesis of a large class of physiologically active
secondary metabolites, phenylpropanoids such as flavonols, isoflavonoids, lignins and
anthocyanins derived from phenylalanine catalyzed through the action of PAL (Weisshaar
& Jenkins, 1998). In the present study imazethapyr mediated increased PAL and POD
activity, consequent increase in phenolic acid and flavonoid contents in lentil seedlings
indicates some sort of secondary metabolism-associated mechanism activated for
quenching of free radical generated through this herbicide upon its exposure.
Phenolic acid and flavonoid content in lentil
Phenolic compounds are widespread plant secondary compounds that play an important
role during ecological imbalances (Curir et al., 1990). This class of biomolecules are
involved in diverse processes like rhizogenesis, vitrification, redox reactions, and stress
resistance (Takahama & Oniki, 1992). In the present investigation increasing phenolic acid
and flavonoid content in lentil seedlings with increasing imazethapyr dose suggested the
major role played by both of the metabolites in regulating the redox balance of the cell.
The biological utility of the phenolic compounds is obtained through their involvement
in the oxidation-reduction process (Narwal, Kumar & Verma, 2016). Enhanced activity of
the POD at higher doses substantiates the active participation of phenolic compounds in
the redox process by providing the substrate for their further oxidation. In this way,
oxidative stress imposed through imazethapyr in lentil seedlings can be neutralized.
Synthesis of the phenolic compounds in plants can be primarily regulated and influenced
through various chemical stimuli induced due to an unfavourable environment that is
perceived by the plants. Herbicide application leads to cellular stress and is associated with
changes in the concentration of the phenolic compounds in plants. Increase in the level of
these compounds were reported in response to some herbicide, while others showed
declining effect (Hoagland, 1990). The current study documented noteworthy differences
in the mean shoot (seedlings) phenolic acid and flavonoid content across different
treatments. Moreover, imazethapyr stimulated the accumulation of phenol in lentil, which
is further noticeable at a higher dose (2 RFD). Similarly, higher phenol accumulation was
also reported in alachlor-treated soybean and maize (Alla & Younis, 1995), oat seedling
treated with glyphosate (Falco, Vilanova & Segura, 1989), acifluorfen-treated spinach
(Kömives & Casida, 1982) and alachlor sprayed sorghum (Molin, Anderson & Porter,
1986). Thus forth, Imazethapyr forms a prototypical representative in the expanding
catalogue of herbicides that can moderate phenol metabolism as well as its accumulation in
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 13/22
lentil seedlings. Earlier studies indicate that herbicides trigger the rate of ROS formation
(Yin et al., 2020). Additionally, increased phenol buildup in response to herbicides gives
plants the ability to detoxify these species produced by free radicals. Basically, the plant’s
use of phenol in its detoxification process suggests a non-enzymatic form of reaction.
Additionally, it offers the starting point for lignin biosynthesis. The level of phenolics,
which is increased when alachlor is applied, controls the growth of soybean and maize
seedlings, illuminating the influence of phenolics on growth behaviour when herbicides are
applied (Alla & Younis, 1995). Imazethapyr is known to retard growth by inhibiting ALS
leading to reduced synthesis of BCAAs and formation of protein. Although, it triggers the
phenol accumulation rate in a dose-dependent manner. Thus, the dose-dependent
accumulation of phenol in response to different imazethapyr treatments in the current
study appears to form a component of the plant defense machinery, which eventually
minimizes oxidative stress generated by the herbicide.
Antioxidant activity in lentil
Phenolic compounds have been illustrated to modulate several biological processes that
include antioxidant activity as well (Kähkönen et al., 1999). The edible as well as non-edible
part of plants is the common source of these compounds (Heim, Tagliaferro & Bobilya,
2002). Earlier studies suggested the inherent capacity of plants gets activated upon
herbicide exposure to defend against the generated consequences through their defense
machinery (Seneff, Swanson & Li, 2015). In our study increased antioxidant activity in
lentil seedlings upon imazethpyr application indicates the activation of these defense
mechanisms through enhanced activity of PAL and POD and increased synthesis of
phenolic acid and flavonoids. Numerous studies indicated a robust positive correlation
between phenol concentration and associated antioxidant activity (Kähkönen et al., 1999).
Herbicides that enhance phenol accumulation in plants, also lead to consequent
stimulation of antioxidant activity (Seneff, Swanson & Li, 2015). Increased antioxidant
activity in lentil seedlings in the present study at higher doses suggests the potential role of
phenolic compounds in overcoming the herbicidal effects. Imazethapyr-induced phenolic
levels exhibited higher antioxidant activity in mung bean and demonstrated a positive
relation between both traits (Namrata et al., 2020). However, reports on high antioxidant
activity exhibited under in vitro systems suggested that there is a meagre chance to combat
ROS in vivo (Halliwell, 1999;Yin et al., 2020). Herbicide stress, similar to other biotic and
abiotic stresses, creates an imbalance in energy between those received and processed by
plants (Tuladhar, Sasidharan & Saudagar, 2021). This inequity usually creates photo
inhibition, ROS formation, and a decline in growth capacity, consequently activating or
accelerating cell death (Tripathy & Oelmüller, 2012). Plants have developed a separate
mechanism for the dissipation of these surplus amounts of energy during its due course
entry in the electron transport chain component of photosynthetic machinery (Havaux &
Kloppstech, 2001;Asada, 1999). It has been reported that under suboptimal conditions, the
diversion of carbon flow shifted to secondary metabolism instead of primary, which leads
to the synthesis of phenolic compounds. These compounds act as energy escape valves
(Hernandez & Van Breusegem, 2010) by dissipating excess energy as fluorescence.
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 14/22
Henceforth, antioxidant activity enhancement under both the DPPH and FRAP assay in
the present study illustrates the involvement of the enzymatic and non-enzymatic
antioxidant system machinery in tandem to overcome the imazethapyr-induced stress.
GR and GST activities in lentil
Glutathione-S-transferase, is another candidate enzyme involved in the detoxification or
inactivation of numerous substrates, including xenobiotic chemicals such as herbicides by
forming conjugates with glutathione. The xenobiotic and the reactive thiol group of
cysteine residue of GST are connected by this process. Furthermore, conjugates are
transported to the vacuoles where detoxification is completed as glutathione conjugates are
hydrolyzed. Besides, it is an important role in detoxification; compartmentalization as well
as chelation of the major toxic material in plants (Anjum et al., 2015), GSTs successively
establish a proficient defense system for plants to defend the ROS-generated effects.
Herbicide selectivity among the weed and crop species depends on the tolerance of the
plant, which is associated with differential routes and rates of herbicide metabolism.
Generally, detoxification involves three series of steps, conversion (step-1), conjugation
(step-II) and deposition (step-III). The coordinated action of all these phases/steps can
detoxify the herbicides with ample speed. Further, accumulation as well as the persistence
of these herbicides to phytotoxic levels are limited through this concerted mechanism.
The increase in GST activity in response to increasing imazethapyr dose and sampling
hour in our study suggests that all these three mechanisms worked in a fine tune to
detoxify/minimize the harmful effects. The greater accumulation of metolachlor in
soybean than in corn is shown to be related to greater herbicide-induced GST activity in
corn than in soybean (Scarponi, Alla & Martinetti, 1992). The different isoforms of
glutathione-s-transferase viz., GST (ALA), GST (CDNB), and GST (MET) get inhibited in
maize treated with isoproturon, while GST (ATR) activity is unaffected, this inhibition was
pronounced at a higher dose of isoproturon (NematAlla, Hassan & El-Bastawisy, 2008).
Thus, maize is subjected to isoproturon-induced oxidative stress, and the extent of this
oxidative damage increases at higher doses with increasing time. In our present study,
imazethapyr induced a dose-dependent increase in the activity of GST compared well with
the report of Shivani et al. (2022), who reported a considerable enhancement in the GST
activity in imazethapyr-treated lentil plants. Zabalza et al. (2007) reported a progressive
increase in the GR activity following imazethapyr treatment in peas from day one of the
treatment. Furthermore, the pronounced increase in GR activities at all the respective
doses throughout the assay period in our study confirms the zabalza et al report in terms of
the rapid impact of imazethapyr on plant and consequent response. Stimulation in GR
activity is also observed with aciflurfen (Hameed et al., 2014) and this enhanced activity of
GR prohibited both oxidation (esp. SH-containing compounds) and lipid peroxidation. A
robust decline was noticed in both glutathione content and activity of GR following the
acifluorfen treatment in the presence of light in cucumber disks (Kenyon & Duke, 1985).
In general, the sensitivity of plants against herbicides appears to be reliant on numerous
factors, like species in practice, the adequate reaction of the plants under a peroxidative
environment (Schmidt & Kunert, 1986), and the metabolism of herbicides in plants (Soares
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 15/22
et al., 2019). The enhancement in antioxidant compound production such as glutathione
and ascorbic acid exhibits the primary response against the herbicide, aciflurfen-induced
peroxidation was noticed in higher plants. This enhanced level of the compounds further
stimulates the GR activity with the concurrent drop in acid-soluble thiol compounds and
the pace of lipid peroxidation (Schmidt & Kunert, 1986). Miteva, Ivanov & Alexieva (2010)
observed that glyphosate treatment provoked an increase in both total and oxidized
glutathione in pea plants and caused activation of GR in treated organs. Enhanced thiol
compounds at higher imazethapyr concentrations in our study are probably the basis for
stimulation in GR activity. These enhancements of the thiol compound at varying
imazethapyr dose explain the abundance of substrate as well as different isoforms of GR
stimulation/activation.
Thiol content in lentil
The increased level of thiol content across the treatment over the control in the present
study ranged from It 31.77% to 74.58%. Moreover, thiol content in different sampling
periods increased progressively with time. Similarly, Aly & Mohamed (2012) also reported
a similar increase in thiol content in maize against metal ion stress. Although, the primary
product in sulphate assimilation is cysteine (Finnegan & Chen, 2012), but glutathione is
reported to be the major thiol compound of the plant cell (Smirnoff, 1993), which provides
plant defense against various stresses (Niu & Liao, 2016). Thiol compound can alternate
between oxidized and reduced states, thus determining the redox status of cell. Plants
grown under sub-optimal environmental conditions usually experience oxidative stress
that leads to an elevated level of ROS (Foyer et al., 1997). Damage caused by ROS is
prevented by various antioxidant molecules such as ascorbic acid, phenolics and thiol
compounds, particularly glutathione with their direct involvement or being a substrate of
enzymes such as GR and GST in the present investigation to detoxify/neutralize the
imazethapyr-associated negative consequences. The role of ascorbic acid and GSH to
minimize the ROS load in cell during oxidative stress in plants is well established
(Hasanuzzaman et al., 2012). In our present study increased thiol content in response to
increasing imazethapyr dose, sampling hour and their interaction advocates the major role
of these thiol compounds to defend the plant against these xenobiotic classes of molecules.
The synchronized actions of these antioxidants are manifested with their involvements as
substrates of APX (ascorbate peroxidase) and GR in the glutathione-ascorbate cycle
(Foyer-Halliwell-Asada pathway) in the protection of cells from ROS-induced toxicity
(Halliwell, 1999).
CONCLUSIONS
Lentil is a nutritious cool-season legume crop that contains a high amount of protein.
The initial growth of the crop is hampered by the massive invasion of weeds. Though there
are several methods to control weeds, the manna generated by herbicides is highly effective
and most preferred. The current experiment aimed to determine how imazethapyr affected
the metabolism of phenol and glutathione as well as the antioxidant behaviour of the lentil
seedlings at various sampling hours after its application. We found that the application of
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 16/22
imazethapyr enhanced the phenolic acid and flavonoid content of lentil seedlings.
Additionally, the antioxidant activity of the phenolic extract in both the DPPH as well as
FRAP assay was also increased. In accordance with an increase in the phenolic
compounds, there was a corresponding increase in mean PAL and POD activity observed,
indicating the modulation of both these enzyme activities in response to herbicide
treatment. The mean thiol content also increased significantly with increasing herbicide
treatments, which appeared to result from an increase in the dose-dependent GR activity.
Based on our findings, it can be summarized that lentil overcome the herbicide-induced
oxidative stress by stimulation of PAL enzyme while detoxifying the parent molecule by
stimulation of GST activity. Consequent upon increased PAL activity, phenolic acid and
flavonoid content and antioxidant activity enhanced indicating the major role played by
phenolics and flavonoids to overcome herbicide-induced stress.
ACKNOWLEDGEMENTS
We are grateful to ICAR-Indian Institute of Sugarcane Research for the approval of study
leave to complete the Doctoral research.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This research was funded by Bidhan Chandra Krishi Viswavidyalaya, Nadia, WB, India
and Researchers Supporting Project number (RSP2023R347), King Saud University,
Riyadh, Saudi Arabia. The Ministry of Education, Youth and Sports of the Czech Republic
(S grant of MSMT CR) supported the APC of this article. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
Bidhan Chandra Krishi Viswavidyalaya: RSP2023R347.
Ministry of Education, Youth and Sports of the Czech Republic: MSMT CR.
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Rajeev Kumar conceived and designed the experiments, performed the experiments,
analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the
article, and approved the final draft.
V. Visha Kumari conceived and designed the experiments, analyzed the data, prepared
figures and/or tables, authored or reviewed drafts of the article, and approved the final
draft.
Ranjit Singh Gujjar conceived and designed the experiments, prepared figures and/or
tables, authored or reviewed drafts of the article, and approved the final draft.
Kumar et al. (2024), PeerJ, DOI 10.7717/peerj.16370 17/22
Mala Kumari conceived and designed the experiments, prepared figures and/or tables,
authored or reviewed drafts of the article, and approved the final draft.
Sanjay Kumar Goswami conceived and designed the experiments, authored or reviewed
drafts of the article, and approved the final draft.
Jhuma Datta conceived and designed the experiments, authored or reviewed drafts of the
article, and approved the final draft.
Srikumar Pal conceived and designed the experiments, authored or reviewed drafts of
the article, and approved the final draft.
Sudhir Kumar Jha conceived and designed the experiments, authored or reviewed drafts
of the article, and approved the final draft.
Ashok Kumar conceived and designed the experiments, authored or reviewed drafts of
the article, and approved the final draft.
Ashwini Dutt Pathak conceived and designed the experiments, authored or reviewed
drafts of the article, and approved the final draft.
Milan Skalicky performed the experiments, analyzed the data, prepared figures and/or
tables, authored or reviewed drafts of the article, and approved the final draft.
Manzer H. Siddiqui performed the experiments, analyzed the data, prepared figures and/
or tables, authored or reviewed drafts of the article, and approved the final draft.
Akbar Hossain performed the experiments, analyzed the data, prepared figures and/or
tables, authored or reviewed drafts of the article, and approved the final draft.
Data Availability
The following information was supplied regarding data availability:
The raw data are available in the Supplemental File.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.16370#supplemental-information.
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