Journal Pre-proof Salt induced modulations in antioxidative defense system of Desmostachya bipinnata Hina Asrar, Tabassum Hussain, Muhammad Qasim, Brent L. Nielsen, Bilquees Gul, M. Ajmal Khan PII:
To appear in:
Plant Physiology and Biochemistry
Received Date: 2 June 2019 Revised Date:
9 December 2019
Accepted Date: 10 December 2019
Please cite this article as: H. Asrar, T. Hussain, M. Qasim, B.L. Nielsen, B. Gul, M.A. Khan, Salt induced modulations in antioxidative defense system of Desmostachya bipinnata, Plant Physiology et Biochemistry (2020), doi: https://doi.org/10.1016/j.plaphy.2019.12.012. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.
Salt induced modulations in antioxidative defense system of Desmostachya
Hina Asrar1┼, Tabassum Hussain1┼, Muhammad Qasim1, Brent L. Nielsen2
Bilquees Gul1*, M. Ajmal Khan1
Institute of Sustainable Halophyte Utilization, University of Karachi, Karachi-75270,
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Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah 84602, USA
Authors contributed equally to the manuscript.
13 14 15 16 17 18 19 20 21 22 23 24
Tel.: (9221) 99261032, Fax (9221) 99261340; e-mail: [email protected]
Declarations of interest: none
This study addressed the interactions between salt stress and the antioxidant responses
of a halophytic grass, Desmostachya bipinnata. Plants were grown in a semi-
hydroponic system and treated with different NaCl concentrations (0 mM, 100 mM,
400 mM) for a month. ROS degradation enzyme activities were stimulated by
addition of NaCl. Synthesis of antioxidant compounds, such as phenols, was
enhanced in the presence of NaCl leading to accumulation of these compounds under
moderate salinity. However, when the ROS production rate exceeded the capacity of
enzyme-controlled degradation, antioxidant compounds were consumed and oxidative
damage was indicated by significant levels of hydrogen peroxide at high salinity. The
cellular concentration of salicylic acid increased upon salt stress, but since no direct
interaction with ROS was detected, a messenger function may be postulated. High
salinity treatment caused a significant decrease of plant growth parameters, whereas
treatment with moderate salinity resulted in optimal growth. The activity and
abundance of superoxide dismutase (SOD) increased with salinity, but the abundance
of SOD isoforms was differentially affected, depending on the NaCl concentration
applied. Detoxification of hydrogen peroxide (H2O2) was executed by catalase and
guaiacol peroxidase at moderate salinity, whereas the enzymes detoxifying H2O2
through the ascorbate/glutathione cycle dominated at high salinity. The redox status
of glutathione was impaired at moderate salinity, whereas the levels of both ascorbate
and glutathione significantly decreased only at high salinity. Apparently, the maximal
activation of enzyme-controlled ROS degradation was insufficient in comparison to
the ROS production at high salinity. As a result, ROS-induced damage could not be
prevented, if the applied stress exceeded a critical value in D. bipinnata plants.
Keywords: salinity, halophyte, oxidative stress, antioxidative enzymes, non-
Desmostachya bipinnata, a C4 perennial grass, belongs to the family Poaceae. It has
high ecological (phytoremediation) and economical (folk medicine and cattle feed)
potential (Pandey et al., 2013; Shaltout et al., 2016). Its distribution in arid and semi-
arid regions of the world has drawn researchers’ interest in investigating its salt
tolerance mechanisms. Such studies will add to our existing understanding and take us
closer to developing salt tolerant crops with improved survival rates. Fulfilment of
this long-desired objective has become even more crucial in the context of more land
becoming saline and the rapidly growing human population.
Exposure of D. bipinnata to saline conditions affects its photosynthetic performance
and, therefore, its growth and development. The restriction in CO2 assimilation
induced at high salinity is associated with an increased dissipation of excitation
energy, damage to PSII reaction center components, decline in reactions of the
Calvin-Benson cycle, and a reduced rate of electron transport (Adnan et al., 2016;
Asrar et al., 2017). Under such conditions, molecular oxygen serves as an alternate
sink for photosynthetic electrons. This results in the formation of reactive oxygen
species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals (Foyer
and Shigeoka, 2011).
The ratio of the electron transport rate to gross photosynthesis increased in D.
bipinnata at high salinity (Asrar et al., 2017). This implies an increased allocation of
electrons to processes other than carbon assimilation. We surmise that C4 plants
suppress photorespiration and provide the photosynthetic electrons with an alternative
pathway in the form of the Mehler reaction (Bräutigam and Gowik, 2016). In
addition, the increased energy demands for cellular processes (ion transport, vacuolar
sequestration, biosynthesis of compatible solutes, etc.) are met with an increased
activity of the mitochondrial electron transport chain. This accelerates the formation
of ROS further (Munns and Tester, 2008). Excessive accumulation of ROS can result
in the execution of cell death. Therefore, strict regulation of their levels is crucial to
ensure the survival of plants (Mittler et al., 2011).
Plants are equipped with antioxidant systems to counter the overproduction of ROS
and avoid or minimize the damage they cause. The enzymatic components includes
antioxidant enzymes such as superoxide dismutase or SOD (EC 220.127.116.11), ascorbate
peroxidase or APX (EC 18.104.22.168), catalase or CAT (EC 22.214.171.124), guaiacol peroxidase
or GPX (EC 126.96.36.199), and glutathione reductase or GR (EC 188.8.131.52). The non-
enzymatic components, on the other hand, consist of hydrophilic (ascorbate and
glutathione) and lipophilic (tocopherols and carotenoids) compounds (Foyer and
Noctor, 2005). Biosynthesis and the activity of the antioxidant system increase under
stress to stabilize the redox balance (Abogadallah, 2010). Many reports show a
positive correlation between efficient antioxidants and the salinity tolerance of plants
(Hamed et al., 2007, 2014; Bouchenak et al., 2012; Benzarti et al., 2014). Exposure to
moderate or high concentrations of NaCl revealed a significant contribution by
proteins related to the antioxidative / redox homeostasis in D. bipinnata (Asrar et al.,
2017). Many proteins were up-regulated or specifically induced to combat a high
salinity-induced oxidative load. Other proteins increased at moderate salinity, i.e.,
apparently in the absence of oxidative stress (as indicated by the values for MDA,
electrolyte leakage and ETR/Ag ratio). The results we obtained highlight the
importance of an ROS-antioxidant interface to maintain physiological metabolism and
stimulate acclamatory responses in plants.
ROS scavenging and its implications in redox homeostasis have been highlighted in
the past decades (Mullineaux and Baker, 2010; Koyro et al., 2013; Demidchik, 2015).
However, studies specifying the relative contribution of enzymatic and non-enzymatic
antioxidants towards salt tolerance are few in number. Therefore, the subject demands
more research. The components of an antioxidant defense system vary from plant to
plant (Ksouri et al., 2007; Souid et al., 2016). Further investigation aimed at
determining the activities of endogenous antioxidants under saline conditions would
be useful. Chief among the potential benefits would be determining which
antioxidants should be adopted as markers to develop salt tolerant crops. Such a result
could have important implications in agro-food biotechnology (Flowers and Muscolo,
A previous study on D. bipinnata revealed that its salt tolerance is based, at least
partly, on its ability to boost the antioxidative defense response (Adnan et al., 2016).
Several antioxidants are involved in keeping the ROS below toxic levels. We were
interested in carrying out an in-depth analysis of the antioxidative defense system to
search for an answer to the following question. Which components can be used as the
markers of stress tolerance in this halophyte?
Therefore, we determined the contribution of various components that were not
considered in previous studies, such as non-enzymatic antioxidants, antioxidant
substrates, and SOD isoforms. Additionally, the total antioxidant capacities of plants
were evaluated to understand the antioxidant system of D. bipinnata.
Thus, the following questions were specifically investigated:
1. Is there a correlation between the abundance of ROS scavenging enzymes and
intensity of salt stress (applied NaCl concentration)? 2. Do all iso-enzymes of SOD respond to salt stress in the same manner?
126 127 128 129
3. Do all phenolic compounds respond in the same way to the changing degrees of NaCl stress? 4. Does the antioxidant system function sufficiently well to protect the plant from ROS stress?
2. Materials and methods
2.1. Plant material and experimental conditions
Seeds of D. bipinnata were germinated in a 1:1 mixture of garden soil and manure in
the growth chamber at 30/20 °C day/night cycle and a photoperiod of 16 h. Three-
week-old seedlings were transferred to pots (6 ×10 cm; height × diameter) in a wire
mesh greenhouse and grown under ambient conditions (temperature: 30 ± 2 °C,
relative humidity: 40 ±10%, PAR: 370 ±50 µmol m−2 s−1). They were watered with
half-strength basic nutrient solution (Epstein, 1972). After six weeks, the seedlings
were transferred to pots (18×25 cm; height × diameter) containing Quartz sand. The
pots were placed in a semi-hydroponic Quick Check System (QCS, Koyro, 2006).
The ambient conditions were 37 ± 4 °C: 47 ± 12 % RH and 1200 ± 200 µmol m−2 s−1
After 2 weeks of acclimation, we treated the plants with solutions of various NaCl (0,
100, and 400 mM) concentrations, termed control, moderate, and high levels of
salinity, respectively. Preliminary experiments were performed to determine the
suitable salinity levels. Ten pots (one plant/pot) were used for each salinity treatment
using a randomized complete block design. The salinity of the nutrient solutions was
increased gradually by adding 50 mM NaCl per day until the desired concentrations
were attained. Solutions were changed every 2 weeks to maintain the nutrient levels.
The duration of the NaCl treatment was 4 weeks.
Fresh weights (FW) of shoot and roots were measured immediately after harvest. For
the dry weight (DW) estimation, the shoot and roots were oven-dried at 60 °C for 72
h and then weighed. Dried plant material was burned in a furnace at 550 °C for 5-7 h
to obtain ash (inorganic content). The organic content was calculated by subtracting
ash content from total dry weight.
Leaf area was calculated with the help of a portable leaf area meter (ADC Bio-
Scientific Ltd. AM350, England). Specific leaf area (SLA) was calculated as the mean
leaf area per unit of leaf dry mass. Leaf relative water content (RWC) was determined
with the procedure reported by Barrs and Weatherley (1962). Leaves (0.5 g) were left
immersed in distilled water at 4 °C overnight. The leaves were blotted dry and their
turgid weight (TW) noted. To obtain the dry weight (DW), the leaves were dried for
48 h at 60 °C. The following formula was used for the RWC calculations:
RWC (%) = (FW – DW) / (TW – DW) ×100
The relative decrease in plant biomass (RDPB), relative leaf area ratio (RLAR), and
salt stress tolerance index (STI) were calculated by using the following equations
(reviewed by Negrao et al. 2017):
RDPB = FWC –FWS / FWC ; RLAR = LARS / LARC ; STI = DWS / DWC
(The subscripts ‘C’ and ‘S’ indicate control and saline treatments, respectively).
2.3. Determination of H2O2 content
Hydrogen peroxide (H2O2) content in D. bipinnata leaves was measured according to
Jessup et al., (1994). Fresh leaf tissue (0.25 g) was homogenized with 5 mL of 3%
ice-cold trichloroacetic acid (TCA) and centrifuged at 12,000 x g, 4 °C for 15 min.
Two mL of TCA extract (supernatant) was mixed with 1 mL of 0.5 M potassium
iodide (KI). The absorbance was recorded at 390 nm (Beckman-Coulter DU-730, UV-
VIS spectrophotometer). H2O2 concentration was estimated with reference to a
standard curve for 0-500 µM H2O2.
2.4. Photosynthetic pigments
Pigments were extracted from leaf tissue (100 mg) in 80% acetone at 4 °C. Cellular
debris was removed by centrifugation at 3500 x g for 5 min at 4 °C. The contents of
pigments were measured by spectrophotometry, according to the equations of
2.5. Determination of ՓPSII and ՓCO2
The effective photochemical quantum yield of PSII (ՓPSII) and the quantum efficiency
of CO2 assimilation (ՓCO ) were measured on a matured fully emerged leaf at
saturating PPFD values for the respective salinity treatments. The ՓPSII was measured
using a pulse modulated chlorophyll fluorimeter (2500 PAM, Walz, Germany) with
the following expression as described by Genty et al., (1989):
ՓPSII = (F'm - Fs)/F'm
where F'm and Fs are maximal and steady-state fluorescence of light-adapted leaves.
The ՓCO was measured with a portable photosynthetic system (LICOR-6400,
Lincoln, NE, USA), according to (Stirling et al., 1991):
ՓCO = A/PPFD
where A represents the rate of CO2 assimilation and PPFD is photon flux density on
the leaf. We express the ratio of ՓPSII and ՓCO as a stress indicator, pointing to a
discrepancy in the electron transfer photochemistry (Fryer et al., 1998).
2.6. Antioxidative enzymes activities
Fresh leaf sample (500 mg) was ground to fine powder with liquid N2 and
homogenized with 5 mL of extraction buffer (50 mM potassium phosphate buffer, pH
7.0, 2% (w/v) polyvinylpolypyrrolidone, 1 mM L-ascorbic acid, and 5 mM disodium
EDTA) in a chilled mortar and pestle. The homogenate was centrifuged at 4 °C for 20
mins at 12,000 x g. The supernatant was used to determine antioxidant enzymes
activity of catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase
The extraction procedure for superoxide dismutase (SOD) and glutathione reductase
(GR) was similar to that mentioned above. The only difference was in the pH of
buffer, i.e., 7.8. Protein concentration was determined, according to Bradford (1976),
using bovine serum albumin as a standard.
Catalase (CAT) activity (ξ = 39.1 mM cm-1) was examined according to Aebi (1984).
The enzyme extract (100 µL) was added to 3 mL of the reaction mixture, containing
potassium phosphate buffer 50 mM (pH 7.0) and 25 mM H2O2. The decreased
absorbance due to the disappearance of H2O2 was recorded at 240 nm.
Ascorbate peroxidase (APX) activity (ξ = 2.8 mM cm-1) was measured by monitoring
the decrease in absorbance due to the oxidation of ascorbic acid at 290 nm (Nakano
and Asada, 1981). The reaction mixture consisted of 50 mM potassium phosphate
buffer (pH 7.0), 0.2 mM EDTA, 0.5 mM ascorbic acid, 2 mM H2O2, and 100 µL
Activity of guaiacol peroxidase (GPX) (ξ = 26.6 mM cm-1) was calculated according
to Zaharieva et al., (1999). A reaction mixture containing potassium phosphate buffer
50 mM (pH 7.0), 2.5 mM H2O2, 2.7 mM guaiacol, and 100 µL enzyme extract was
prepared. The increase in the absorbance due to formation of tetra-guaiacol was
measured for 1 min at 270 nm.
Glutathione reductase (GR) activity (ξ = 6.2 mM cm-1) was measured according to
Halliwell and Foyer (1978). The enzyme extract (50 µL) was added to a reaction
mixture containing 100 mM Tris-HCl (pH 7.8), 5.16 mM EDTA, 0.31 mM NADPH,
and 0.51 mM oxidized glutathione (GSSG). The decrease in absorbance due to
oxidation of NADPH was recorded at 340 nm and used to calculate the activity.
The superoxide dismutase (SOD) activity assay was based on the principle of the
photochemical reduction of nitro blue tetrazolium (NBT), as described by Beyer and
Fridovich (1987). The enzyme extract (40 µL) was added to a reaction mixture
containing 0.05 mM NBT, 10 mM L-methionine, 0.22% Triton X-100, and 0.12 mM
riboflavin in 50 mM potassium phosphate buffer (pH 7.8). One set of test tubes
containing the reaction mixture was kept under a 40 W fluorescence light. Another set
was placed in the complete dark for a period of 7 minutes. The increase in absorbance
at 560 nm due to the formation of formazan under light was measured against the
control, i.e., the test tube placed in the dark. The absorbance recorded in the absence
of enzyme extract was taken as 100%. Enzyme activity was calculated as the
percentage inhibition per min.
2.7. SOD isozymes
Native polyacrylamide gel electrophoresis (PAGE) was carried out according to
Laemmli (1970) on 12% polyacrylamide slab gels, using a Mini-PROTEAN Tetra
cell (BioRad, Hercules, CA, USA). 40 µg of protein extract was loaded in each gel
lane. The activity of SOD was visualized by a photochemical NBT reduction method
(Beauchamp & Fridovich, 1971). Different isoforms of SOD were identified by
separate incubation of gels in the staining buffer (50 mM potassium phosphate buffer,
pH 7.8, 0.1 mM EDTA, 28 mM TEMED, 0.003 mM riboflavin and 0.25 mM NBT),
either with 5 mm H2O2 or 2 mM potassium cyanide (KCN) (Salin and Bridges, 1980).
KCN and H2O2 inhibit CuZn-SOD activity while H2O2 inhibits that of Fe-SOD. Mn-
SOD is not inhibited by either KCN or H2O2.
The gels were immediately scanned with the GenoSens gel documentation system
(Clinx Science Instruments). Images were analyzed to estimate the relative intensity
of each band with CIS 1 D analysis software (Clinx, GenoSens Series, Gel
documentation system). The intensity of bands from the control treatment was taken
as a reference and made = 100.
2.8. Antioxidants substrates and the determination of their redox proportions
The ascorbate content as reduced (ASC) and total ascorbate [ASC+oxidized ascorbate
(DHA)] was determined according to Kampfenkel et al. (1995). The reaction mixture
(4 mL) for measuring the ASC content consisted of leaf TCA extract (6% TCA), 30
mM potassium phosphate buffer (pH 7.4), 2.5% TCA, 8.4% orthophosphoric acid
(H3PO4), 0.8% bipyridyl, and 0.3% ferric chloride (FeCl3). After incubation at 42°C
for 40 min in a water bath, the absorbance of the test solution was recorded at 525 nm
(Beckman-Coulter DU-730, UV-VIS spectrophotometer).
For measuring the total ascorbate (ASC+DHA) content, the additional steps included
the reduction of DHA to ASC by incubation with 0.5 mM dithiothreitol (DTT) at
42°C for 15 min, and then the removal of excess DTT with 0.025% N-ethylmaleimide
(NEM). The contents of oxidized (DHA) and total ascorbate (ASC+DHA) were
estimated with reference curves of dehydroascorbic acid and L-ascorbate solutions,
respectively. The concentration of reduced ascorbate (ASC) was calculated by
subtracting that of DHA from total ascorbate. The ratio of oxidized and reduced
ascorbate was also calculated.
Glutathione content, as reduced (GSH) and total glutathione [GSH+ oxidized
glutathione (GSSG)], was determined according to Anderson (1985) with some
modification. The reaction mixture for measuring GSH content consisted of leaf TCA
extract (3% TCA), potassium phosphate buffer (pH 7.4) and 50 mM, containing 0.5
mM ethylene diamine tetraacetic acid (EDTA) and 0.005% 5,5-dithiobis-(2-
nitrobenzoic acid) (DTNB). Absorbance was measured at 412 nm after keeping the
reaction mixture at 30°C for 2 min.
Total glutathione (GSH+GSSG) content was determined after the reduction of GSSG
to GSH by adding 0.2 mM nicotinamide adenine dinucleotide phosphate (NADPH)
and 130 mM potassium phosphate buffer (pH 7.4) containing one unit of glutathione
reductase (GR). The reaction mixture was incubated at 30°C for 30 min to allow a
reaction between the enzyme and the substrate. Absorbance was measured at 412 nm
(Beckman-Coulter DU-730, UV-VIS spectrophotometer) and the contents of GSH
and GSH+GSSG were estimated with standard curves of glutathione (GSH) solutions
as reference. The content of oxidized glutathione (GSSG) was calculated by
subtracting GSH from total glutathione. The ratio of reduced to oxidized glutathione
2.9. Antioxidant capacity assays
The antioxidant capacity of D. bipinnata was determined using 1,1-Diphenyl-2-
picryl-hydrazyl (DPPH; Brand-Williams et al., 1995) and 2,2'-azino-bis3-
ethylbenzothiazoline-6-sulphonic acid (ABTS; Re et al., 1999) radical scavenging
tests. The reducing potential was estimated with the ferric reducing antioxidant power
assay (FRAP; Benzie and Strain, 1996) and total antioxidant capacity using
phosphomolybdate complex method (TAC; Prieto et al., 1999).
2.10. Determination of total phenolic (TPC), flavonoid (TFC), proanthocyanidin
(PC), and tannin (TTC) contents
Total phenolic content (TPC) was determined using the Folin-Ciocalteu colorimetric
method (Singleton and Rossi, 1965). Colorimetric methods were also used to quantify
total flavonoids (TFC; Chang et al., 2002), total proanthocyanidin (PC; Sun et al.,
1998), and total tannins (TTC; Pearson, 1976).
2.11. High-performance liquid chromatography (HPLC) analysis
Plant samples (0.5 g) were extracted in 40 ml aqueous methanol and 10 ml HCl
(Proestos et al., 2006). Extracts were sonicated (15 min) after purging nitrogen (60 s),
and then refluxed in a boiling-water bath (120 min). Afterward, the extracts were
filtered, methanol was added (up to 100 ml), and filtered again (0.45 µm membrane
filter, Millex-HV) before being injected onto a HPLC system.
The HPLC system (Shimadzu LC-20AT) included an auto-sampler (SIL-20A), a
photo-diode array detector (SPD-M20A), column oven (CTO-20A), Nucleosil C18
column (5 µm 100A, 250 x 4.60 mm, Phenomenex), guard column (KJO-4282,
Phenomenex), and LC Solution software. Sodium phosphate buffer (50 mM; pH 3.3)
and 70% methanol were used as the mobile phase. The volume of the injection was10
µL and the flow rate was maintained at 1.2 ml min-1. The gradient program by
Sakakibara et al., (2003) was used.
2.12. Statistical analyses
The statistical software, SPSS, version 24 (SPSS Inc., Chicago, USA) was used for
analysis, followed by analysis of variance (ANOVA) to test the significance of all
parameters. The Bonferroni test was carried out to determine if significant (p < 0.05)
differences existed among means. In addition, a principal component analysis (PCA)
was performed with the help of SPSS (v. 24) to correlate the measured parameters on
plants exposed to salinity treatments. Every principal component was a linear
combination of original variables with coefficients equal to eigenvectors of the
correlation matrix. Finally, a 2D graphical interpretation of main components was
obtained with the same software.
3.1. Growth parameters
A significant increase in both root FW (67%) and DW (81%) was recorded at
moderate salinity. The shoot biomass (FW and DW) was similar to that of control
plants. In contrast, the high salinity treatment caused considerable reduction in FW
and DW of shoot and roots. A linear increase with increasing salinity in the organic
content of both the shoot and roots of the plants was recorded. A similar trend was
observed for the ash (inorganic) content of shoots. However, in roots, it declined
substantially at 400 mM NaCl.
The RWC was similar (approximately 86%) for all treatments. The maximum value
for the specific leaf area was observed at 100 mM NaCl. It decreased by 31% at 400
mM NaCl, in comparison to the control treatment plants (Fig. 1). The drastic effects
of high salinity on the growth of D. bipinnata were evident from the relative decrease
in plant biomass and relative leaf area ratio. The salt tolerance index of these plants
was also low (Supplementary Table 1). The plants grown in the presence of 400 mM
NaCl showed a lower chlorophyll concentration than those exposed to control and
moderate salinity. (Fig. 2).
3.2. Oxidative stress indicators
A significant increase in the ratio of ՓPSII / ՓCO2 was noted at high salinity. These
results were concomitant with a substantial increase in the content of H2O2, which
was 33% higher as compared to the control plants. In contrast, in 100 mM NaCl14
treated D. bipinnata, the values for ՓPSII / ՓCO2 and H2O2 remained almost constant
3.3. Antioxidant enzyme activities
The 400 mM NaCl-treated D. bipinnata plants showed maximum activities of SOD,
GR, and APX, which increased by 35, 35 and 40% respectively, when compared with
activities of plants from control treatments. The activity showed the highest
correlation coefficient with salinity treatments (r2 = 0.81 for SOD, 0.97 for GR and
0.85 for APX) among all the analyzed enzymatic antioxidants. A transient increase in
the activity of GPX (209%) and CAT (41%) was recorded at moderate salinity,
followed by a decline at high salinity (Fig. 3).
3.4. SOD isoforms
Fig. 4 shows the changes in the various isoforms of SOD in response to the applied
treatments. An analysis of gels revealed an increase in their levels of all SOD
isoforms (Mn-SOD, Fe-SOD, and Cu/Zn-SOD) under salinity with the maximum
value at 400 mM NaCl. The constitutive expression of Fe-SOD2 was high as
compared to that of the other isoforms. The largest change in the expression levels
under salinity was recorded for iron-containing SOD isoforms (Fe-SOD1 and Fe-
3.5. Non-Enzymatic antioxidants: ascorbate and glutathione
An investigation of non-enzymatic antioxidants, i.e., ascorbate, and glutathione and
their respective pools revealed a linear increase in the oxidized forms. DHA and
GSSG levels rose with increasing salinity, but a non-significant change was recorded
for reduced forms, i.e., ASC and GSH (Table 1). Total ascorbate content increased
(20%) at high salinity when compared to the control. However, total glutathione
content showed no significant change. The ASC/DHA ratio was unchanged at 100 15
mM NaCl but at 400 mM it decreased significantly. In contrast, a significant decrease
in the GSH/GSSG ratio was recorded even at moderate salinity. We noted that total
ascorbate showed a high correlation coefficient (r2 = 0.96) with salinity treatments
while the accumulation of glutathione seemed unrelated (r2 = 0.25).
3.6. Antioxidant capacity – ABTS, DPPH, and FRAP methods
The total antioxidant capacity of D. bipinnata leaf extracts did not vary significantly
under salinity treatments (r2 = -0.19). However, an assessment of antioxidant activity
by DPPH, ABTS, and FRAP methods showed an influence of salinity concentration
on these individual tests. Antioxidant activity, based on the capacity of the leaf extract
to scavenge DPPH free radicals, was maximum in plants treated with moderate
salinity (32% more than that of control plants). It showed the lowest values in the
high salinity treatment. Likewise, the highest antioxidant activity evaluated with
ABTS test was recorded for plants given the 100 mM NaCl treatment. It remained
unchanged at 400 mM NaCl when compared to control-treated plants (Fig. 5).
However, the ferric reducing antioxidant potential (FRAP) decreased (approx. 22%)
only at high salinity. It presented a highly negative correlation with salinity treatments
(r2 = -0.85).
3.7. Total phenolic (TPC), flavonoid (TFC), proanthocyanidin (PC), and tannin
The salinity treatments resulted in significant changes in TFC. The highest value was
found at moderate salinity and the lowest one at high salinity (Fig. 6). Total
polyphenol was increased (28%) under moderate salinity. In contrast, PC did
decreased by 41% at high salinity. Total tannin content showed a progressive decrease
with increasing NaCl concentration. We noted the highest negative correlation of
TTC (r2 = -0.97) and PC (r2 = -0.94) with saline treatments.
3.8. Phenol profiling
The phenolic composition of D. bipinnata leaves was determined using HPLC.
Several phenolic compounds were identified, including pyrocatechol, catechin,
chlorogenic acid, caffeic acid, salicylic acid, coumaric acid, coumarin, cinnamic acid,
quercetin, and kaempferol (Supplementary Fig. 1). In general, most phenolic
compounds (pyrocatechol, chlorogenic acid, coumaric acid, coumarin, quercetin, and
kaempferol) significantly increased at moderate salinity. However, the high salinity
treatment caused a reduction in all but four phenolic acids. Those acids either
increased (salicylic and coumaric acids) or remained unchanged (caffeic and cinnamic
acids) (Fig. 7). Among all the phenols identified under salinity treatments, the content
of catechin (> 2 mg g-1 DW) was the highest while that of quercetin (< 0.15 mg g-1
DW) was the lowest. ANOVA revealed a strong positive correlation between
moderate salinity and phenolic compounds (except catechin). However, high salinity
negatively affected phenolic compounds with the exception of salicylic acid (r2 =
0.922) and coumaric acid (r2 = 0.984).
3.9. Principal Component Analysis (PCA)
The first two principal components (PCs) explained 79.10% of the cumulative
variance with PC1 and PC2, contributing 53.7% and 25.4% of the total variance,
respectively (Fig. 8). It is evident that the antioxidant enzymes, total and oxidized
fractions of ascorbate and glutathione, H2O2 levels, ՓPSII/ՓCO2, and some phenols
(coumaric, cinnamic and salicylic acids) were strongly correlated. Therefore, their
responses can be separated from the other measured parameters (FW, chlorophyll,
antioxidant capacity, ASC/DHA, GSH/GSSG, reduced forms of glutathione and
ascorbate, flavonoids, tannins, proanthocyanidin, and most of the phenols) by PC1.
However, PC2 revealed a negative correlation of CAT, GPX, and SOD with the other 17
enzymatic and non-enzymatic antioxidants. A strong negative correlation was also
observed among the antioxidant activity determining methods and GSH, ASC,
ASC/DHA, and GSH/GSSG.
There are two components of salinity stress: ionic stress and osmotic stress (Munns,
2002). As a result, closure of the stomata and an inhibition in gas exchange have been
observed due to limited availability of CO2 (Flowers and Colmer, 2015). Especially in
the presence of high light intensity, this stressful situation becomes even worse due to
increased production of reactive oxygen species (ROS). Their concentration may
reach toxic levels (Foyer and Shigeoka, 2011). ROS production has been described as
a side reaction of photosynthetic activity. Due to their redox potentials, compounds
such as light-activated chlorophyll and reduced ferredoxin can transfer an electron to
molecular oxygen to produce an oxygen radical. The probability of this reaction will
increase if NADP+, the physiological acceptor of photosynthetic electron transport, is
not available. This will be the case if the absorption rate of light quanta significantly
exceeds that of NADPH consumption in photosynthesis (Foyer and Noctor, 2005).
Under optimal growth conditions, the ROS production rate will be balanced by the
rate of their degradation. In many plant species, most of the surplus electrons will be
consumed by the glutathione-ascorbate-cycle as described by Asada (Foyer and
Shigeoka, 2011). In the case of ROS overproduction, the concerted action of
antioxidants and ROS-scavenging enzymes will keep ROS concentrations low. Plant
species differ (i) in their contents of antioxidative system and (ii) in their capacity to
produce antioxidants under stress. This study addressed the responses of D. bipinnata
by comparing the mechanisms involved in oxidative stress tolerance in response to
various NaCl treatments.
4.1. Effects on plant growth
In agreement with the previous reports on D. bipinnata (Asrar et al., 2017, 2018) and
other halophytic grasses (Flowers and Colmer, 2015), we observed that growth of D.
bipinnata was stimulated by addition of 100 mM NaCl to the culture medium.
Usually, extensive root growth prevents the accumulation of inorganic ions, mainly
Na+, in the shoots (Munns and Tester, 2008). However, we found this was not the
case in our study, as we found increased ash content in the shoots. The increased
organic content in both shoots and roots under high salinity signifies its contribution
in osmotic adjustment (Flowers and Colmer, 2008). Thus, plants were able to
maintain their leaf RWC under salinity. The observed reduction in SLA and RLAR at
400 mM NaCl indicates the presence of smaller and thicker leaves with fewer
stomata, a strategy to conserve water. This may explain the reduction in transpiration
(Asrar et al., 2017). In addition, a reduction in RLAR frees more tissue volume to
sequester more salts into the vacuoles (Munns, 2002). The damaging effects of toxic
salt concentration are apparent in the low values of STI and RDPB (Supplementary
Table 1). On the other hand, the relative decrease in plant biomass reflects a switched
allocation of available resources from biomass accumulation to energy used in stress-
resisting mechanisms (Lavinsky et al., 2015; Nam et al., 2015).
4.2. Disturbance in photosynthesis leads to ROS production
High values of the Փ PSII/Փ CO2 ratio (Fig. 2) demonstrate the availability of reducing
power (NADPH) in excess of its utilization in the Calvin-Benson cycle. This
restricted regeneration of NADP+ is known to increase the probability of ROS
production (Mehler reaction; Fryer et al., 1998). The increased concentration of H2O2,
found in D. bipinnata leaves (Fig. 2) will cause oxidative damage to membrane lipids,
proteins, and DNA molecules. It also inactivates enzymes and introduces an
imbalance in the cellular redox systems (Mittler, 2002; Hamed et al., 2014). The
decreased chlorophyll content in response to high salinity treatment (Fig. 2) may be
an adaptive strategy to avoid absorption of excessive light. Thereby, limiting ROS
production on the expense of photosynthetic capacity.
4.3. Components of the antioxidant system and their presumptive functions
4.3.1. SOD isoforms
The activity of the antioxidant enzyme system (Fig. 3) demonstrates the potential of
D. bipinnata to minimize the toxic effects of ROS under salt treatment. SOD, acting
as the first line of defense, has been found to increase in several other halophytes
(Amor et al., 2005; Lokhande et al., 2011; Bose et al., 2014; Hussain et al., 2015). We
found this increased activity concomitant with the up-regulation of SOD isoforms
(Figure 4). Among these, the largest increase in the content of Fe-SODs suggests they
play a predominant role in scavenging chloroplastic-O2.- (Myouga et al., 2008). The
increase may also be explained as an adaptive strategy of plants to bind excessive Fe -
- a redox active metal ion with the potential to generate the most damaging ROS (i.e.
OH· ) -- from participation in Fenton reaction. This explanation is intriguing, as a
reduction in the expression of a vacuolar iron-sequestering protein was reported
previously (Asrar et al., 2018). In addition, increased expression of other SOD
isoforms (i.e. Mn-SOD, Cu/Zn-SOD) has been observed. They are found in the
mitochondria, cytosol, and apoplastic regions. This suggests a strategic control of the
plant’s defense system in detoxifying ROS at their places of origin.
Of particular importance is the accumulation of Cu/Zn-SODII, which resides in close
proximity to photosystem I (PSI). Therefore, SOD may actively scavenge ROS
generated in this region (Kliebenstein et al., 1998). This is quite interesting, as our
previous work proposed salinity-induced damage in the PSI electron transport chain 20
(Asrar et al., 2017). However, the results we obtained in this study are in contrast to
those reported for some salt sensitive plants.
In pea plants, an increased expression of one SOD isoform is accompanied by
inhibited expression of other isoforms (Hernandez et al., 1995; Gomez et al., 1999).
This may characterize an important difference of the salt tolerance mechanism of C3
plants (such as pea) and our test species, which is a C4 plant. Apparently it is
important for this plant’s type of C4 pathway to efficiently scavenge ROS produced in
bundle sheath chloroplasts as well as in mitochondria.
Summarily, the differential accumulation of the various isoforms of enzymatic
antioxidant SOD enhances the tolerance of D. bipinnata to oxidative stress by
catalyzing the conversion of O2.- into H2O2 at various sub-cellular sites. However, the
contribution of other related antioxidant enzymes and their isoforms in combating
oxidative stress cannot be neglected and should be examined in future studies. These
findings also answer one of the queries of this study: all isozymes of SOD do not
respond to salt stress in the same manner.
4.3.2. Antioxidant enzymes
The enhanced SOD activity led to increased H2O2 content causing secondary
oxidative stress in our test species. Similar observations have been reported for other
halophytes as well (Amor et al., 2005; Lu et al., 2016). Enzymes such as CAT, APX,
GPX, and GR, among others, regulate the levels of H2O2. The considerably high
activities of APX and GR indicated the involvement of the ASC/GSH cycle to
scavenge H2O2 under high salinity treatment. On the other hand, CAT and GPX were
involved in the detoxification of H2O2 mainly at moderate salinity. This observed
correlation between applied NaCl concentrations and the abundance of ROS-
scavenging enzymes answers our first question in this study. As a response to salt
stress, the redox state of the glutathione pool became more oxidized (Table 1).
Increased concentrations of GSSG will enhance the regeneration of NADP+. The
competition for electrons will reduce ROS formation, as discussed by Foyer and
4.3.3. The ascorbate-glutathione cycle
In the presence of moderate salinity, we observed in our test species, an unchanged
ASC/DHA ratio (Table 1). Previously, a low value for MDA and electrolyte leakage
was recorded in response to similar treatment (Asrar et al., 2017, 2018). Apparently,
ROS production induced by moderate NaCl concentrations can be balanced by the
Asada pathway. Cellular ROS concentration is kept low and therefore damage, such
as peroxidation of membrane lipids, is prevented. From this conclusion, it may be
reasoned that salinity tolerance of D. bipinnata is limited by the capacity of the
ascorbate-glutathione cycle to compensate for ROS over-production and, thus, control
cellular ROS concentration.
Stress induced over-expression of respective genes results in an increased abundance
of the enzymes controlling the above mentioned reactions. While such enzyme
production takes time, ROS detoxification by direct reaction with antioxidants is an
immediate reaction, saving other cell components from peroxidation. For a better
understanding of this part of the ROS defense system, we have used four different
assays, DPPH, ABTS, TAC, and FRAP (Fig. 5). As described in the literature (Melo
et al., 2008; Rodrigues et al., 2011; Berłowski et al., 2013), these tests allow
evaluation of the capacity of a cell extract to detoxify ROS, and measurements of
individual concentrations of some major antioxidants. Our results show that treatment
of D. bipinnata plants with moderate salinity results in the stimulation of antioxidant
capacity and is associated with enhanced cellular concentration of phenols and
flavonoids (Fig. 6). Antioxidants do not undergo a regeneration cycle. Thus, their
concentrations were significantly reduced in the presence of high NaCl stress, when
the ROS production rate exceeded the detoxification capacity of enzyme-controlled
reactions such as the Asada cycle. Results of the correlation analysis indicate that
cellular antioxidative capacity is linked to the concentrations of phenols and
flavonoids. On the other hand, only a low correlation was found with respect to
concentrations of other compounds, such as tannins and pro-anthocyanidin. Thus, we
conclude that the ABTS assay may be a key tool to rank the antioxidative defense
response in related plant species.
Phenols have been shown to improve tolerance to other types of stresses as well, such
as heavy metal stress (Bravo, 1998; de Groot and Rauen, 1998; Simiæ et al., 2007;
Maurya and Devasagayam, 2010). Therefore, we applied HPLC analysis to evaluate
the effects of NaCl on the abundance of several phenolic compounds (Fig. 7).
Increased contents of kaempferol and quercetin have to be interpreted in the context
of their involvement in the biosynthesis of glutathione (Moskaug et al., 2005).
Kampeferol also plays an important role in control of meristematic activities, being a
cofactor of auxin (Janesen, 2002).
Salicylic acid is known to have two functions: it can act as an antioxidant (Simić et
al., 2007) as well as a second messenger signaling ROS-caused stress events
(Pirasteh-Anosheh and Emam, 2018; Kim et al., 2018). While the concentration of
most antioxidants decreased in the presence of 400mM NaCl, salicylic acid
concentration remained at an increased level. In a similar way, the cellular
concentration of coumaric acid was stimulated by addition of NaCl and did not
correlate with the antioxidative capacity of cells. We, therefore, conclude that in D.
bipinnata these two compounds are not involved in ROS detoxification, as found by
Rezazadeh et al., (2012) e. Rather, their role as a pro-oxidant can be presumed. These
analyses allowed us to answer another question: all phenolic compounds did not
respond in the same way to changes in the degree of NaCl stress.
The present study provides information about responses to moderate and high salinity
of the antioxidative defense system of D. bipinnata. We have analyzed plant samples
after an extended period of salt treatment. We have observed different concentrations
of antioxidant compounds and enzymes involved in ROS degradation. Our
interpretation of these observations is summarized in fig. 9.
Our results differ from earlier observations of other research groups (Hernandez et al.,
1995; Gomez et al., 1999). This can because we have used D. bipinnata as an
experimental plant, which employs C4 metabolism for assimilation of CO2, while in
the cited publications stress responses of a C3 plant were analyzed.
A more detailed analysis of biochemical pathways resulting in the observed
differences requires identification of the type of C4 photosynthesis used by D.
bipinnata. From earlier experiments, we could conclude that D. bipinnata is using
PEP carboxykinase type CO2 fixation. However, in accordance with findings of
Schlüter et al. (2016), we speculate that a modification of equilibria between
metabolites can be achieved under stress by tuning activities of metabolic pathways.
Therefore, we currently are unable to describe plant responses to NaCl at the
biochemical level in detail, as we lack information such as measurement of stress
effects on the genetic control of expression of enzymes of the C4 pathway.
In this context, stress-induced changes in the patterns of SOD isoenzymes indicate
that sites of ROS production vary in activity, depending on the degree of salt stress.
As ROS production is an indicator of bottlenecks in metabolism, it may be concluded
that in the presence of 100 mM NaCl and 400mM NaCl different intermediates will
build up. These intermediates may be substrates of different secondary metabolic
pathways. Therefore, we have found different patterns of secondary metabolites in the
presence of different NaCl concentrations.
HPLC analysis of plant extracts showed that salt treatment resulted in increased
cellular salicylic acid concentration. It may be postulated that this acts as a signal that
will stimulate a transcription factor controlling the expression of NaCl-responsive
genes. Thus, salicylic acid may be used as an indicator of NaCl stress, but apparently
it does not tell the degree of stress.
Recognizing moderate stress is possible if we measure concentrations of phenolic
antioxidants consumed by ROS detoxification and present as a proof of concept. If
stress exceeds a threshold value, concentrations of these antioxidants are too low to
allow a reliable ranking of stress degree. In our experiments, this is the case in the
presence of 400 mM NaCl. Our results also allow us to state that the concentration of
antioxidants and the activity of ROS degrading enzymes was sufficient to save the
plant from ROS stress at moderate salinity treatment. These conclusions are
summarized in Fig. 9. As we do not know the exact pathway of C4 photosynthesis in
D. bipinnata, we have shown only one hypothetical cell containing one chloroplast
and one mitochondrion instead of the two cell types involved.
615 616 617 618 619 620 621
Fig. 1. Fresh weight (FW: A and B), dry weight (DW: C and D), ash (E and F) and organic weight: OW (G and H) of shoot and roots, relative water content (I) and specific leaf area (J) in D. bipinnata subjected to different NaCl concentrations. Values represent the mean ± S.E. of three replicates (n = 3). Different letters indicate significant differences due to salt treatments, according to Bonferroni’s test (P < 0.05). 26
Fig. 2. Chlorophyll content (A), ratio between quantum efficiencies of linear electron
624 625 626 627 628
transport through PSII and of CO2 assimilation (ՓPSII / ՓCO2: B), and hydrogenperoxide (H2O2: C) content in leaves of D. bipinnata subjected to different NaCl concentrations. Values represent the mean ± S.E. of three replicates (n = 3). Different letters indicate significant differences due to salt treatments, according to Bonferroni’s test (P < 0.05). 27
630 631 632 633 634 635
Fig. 3. Superoxide dismutase (SOD: A), glutathione reductase (GR: C) ascorbate peroxidase (APX: D), guaiacol peroxidase (GPX: D), and catalase (CAT: E), activity in leaves of D. bipinnata subjected to different NaCl concentrations. Values represent the mean ± S.E. of three replicates (n = 3). Different letters indicate significant differences due to salt treatments, according to Bonferroni’s test (P < 0.05). 28
637 638 639 640 641 642 643 644 645
Fig. 4. Isozymes of superoxide dismutase (SOD) and changes in their contents isolated from leaves of D. bipinnata subjected to different NaCl concentrations. For each lane, 40 µg of protein extract was loaded. Isozymes are present on Coomassie blue-stained SDS-PAGE gels. The values are given as % of control ± S.E.
646 647 648 649 650 651 652
653 654 655 656 657
Fig. 5. Effect of NaCl on antioxidant activities of Desmostachya bipinnata. Values represent the mean ± S.E. of three replicates (n = 3). Different letters indicate significant differences due to salt treatments, according to Bonferroni’s test (P < 0.05).
658 659 660 661 662 663 664 665 666 667 668 669 670 30
671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689
Fig. 6. Effect of NaCl on contents of total polyphenol (TPC), proanthocyanidin (PC) total flavonoid (TFC), and tannin (TTC) in leaf extracts of Desmostachya bipinnata. Values represent the mean ± S.E. of three replicates (n = 3). Different letters indicate significant differences due to salt treatments, according to Bonferroni’s test (P < 0.05).
690 691 692 693 694 695 696 697 698 699 700 701 31
702 703 704 705 706
Fig. 7. Effect of NaCl on the contents of various phenolic compounds in the leaves of Desmostachya bipinnata. Values represent the mean ± S.E. of three replicates (n = 3). Different letters indicate significant differences due to salt treatments, according to Bonferroni’s test (P < 0.05).
709 710 711 712 713
Table 1. Ascorbate- and glutathione- pools in leaves of D. bipinnata subjected to (a) 0 mM NaCl, (b) 100 mM NaCl, and (c) 400 mM NaCl treatments. The values are given as mean ± S.E. Different letters indicate significant differences due to salt treatments, according to Bonferroni’s test (P < 0.05). ). n.s. indicates no significant difference due to treatments.
ASC NaCl -1 (mM) (µmol g FW)
714 715 716 717 718 719 720 721 722 723
(µmol g FW)
Total Ascorbate -1
(µmol g FW)
GSH (nmol g-1 FW)
GSSG Total Glutathione (nmol g-1 FW) (nmol g-1 FW)
2.43 ± 0.07n.s. 4.41 ± 0.20a 6.84 ± 0.15a
0.55 ± 0.02a
233.20 ± 9.76 n.s. 22.72 ± 1.01a 255.91 ± 10.54n.s.
10.28 ± 0.30a
2.17 ± 0.01n.s. 5.14 ± 0.02b 7.31 ± 0.04a
0.42 ± 0.00a
224.65 ±14.16n.s. 38.27 ± 3.70b 259.92 ± 10.95n.s.
5.97 ± 0.95b
2.08 ± 0.12n.s. 6.11 ± 0.10c 8.19 ± 0.09b
0.34 ± 0.02b
210.35 ±10.26n.s. 55.04 ± 0.80c
3.83 ± 0.24b
265.39 ± 9.48n.s.
ASC: ascorbate reduced state; DHA: ascorbate oxidized state; GSH: glutathione reduced state; GSSG: glutathione oxidized state
724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760
Fig. 8. Principal Component Analysis (PCA). Site score plots of the studied variables in the salt stress treatments for D. bipinnata. PCAs included, as analysed variables: total polyphenol (TPC), proanthocyanidin (PC) total flavonoid (TFC), tannin (TTC), ascorbate reduced state (ASC), ascorbate oxidized state (DHA), glutathione reduced state (GSH), glutathione oxidized state (GSSG), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) guaiacol peroxidase (GPX), hydrogen peroxide (H2O2), chlorophyll (CHL), ratio between quantum efficiencies of electron transport and CO2 assimilation (PSIPS2), water content (WC), and antioxidant activities determined by different assays (FRAP, DPPH, ABTS).
761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785
Fig. 9. A model scheme summarizing the physiological and antioxidant responses of Desmostachya bipinnata treated with moderate (100mM) and high (400mM) NaCl. Analyzed parameters after integration to known locations were supported with arrows to highlight response pattern. Arrows (up- or down- head) indicated increase or decrease in a response while left-right arrow indicated an unchanged response, in comparison to that of control plants. Green color arrows represent moderate salinity while red color arrows indicate high salinity treatment. The length of the arrow increased with increasing response difference. Plants treated with moderate salinity were able to mitigate H2O2 due to integrated functioning of several enzymatic and non-enzymatic antioxidants. On the other hand, plants treated with high salinity could not scavenge increased H2O2 mainly because of insufficient contents of non-enzymatic antioxidants and little antioxidant activities displayed. Abbreviations/symbols: ascorbate reduced state (ASC), ascorbate oxidized state (DHA), glutathione reduced state (GSH), glutathione oxidized state (GSSG), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) guaiacol peroxidase (GPX), isozymes of SOD: MnSOD, Cu/ZnSOD, and FeSOD, chlorophyll (Chl), ratio between quantum efficiencies of electron transport and CO2 assimilation (ՓPSII / ՓCO2), antioxidant activities as determined by FRAP, DPPH, and ABTS assays, photosysem (PS), superoxide anion (.O2-). hydrogen peroxide (H2O2), and hydroxyl radical (OH.).
787 788 789 790 791 792
Supplementary Fig. 1. HPLC chromatograms showing phenolic profile (1-hydroquinone, 2gallic acid, 3- resorcinol, 4- pyrocatechol, 5- catechin, 6- chlorogenic acid, 7- caffeic acid, 8salicylic acid, 9- coumaric acid, 10- coumarin, 11- cinnamic acid, 12- quercetin, 13kaempferol and 14- naringenin) of standard compounds and leaf extracts of Desmostachya bipinnata.
793 794 795
Supplementary Table 1. Relative decrease in plant biomass (RDPB), relative leaf area ratio (RLAR) and salt tolerance index (STI) of D. bipinnata under moderate and high salinity treatments. The values are given as% of control ± S.E. NaCl (mM)
-0.09 ± 0.03a
1.01 ± 0.03a
-0.02 ± 0.04 b
0.63 ± 0.83b
796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 37
1. Antioxidant responses of Desmostachya bipinnata to varying concentrations of NaCl were
2. The contribution of enzymatic and non-enzymatic antioxidants varied with the applied
3. Plants were not able to overcome high salinity induced oxidative stress.
4. The growth of the plants was hampered mainly because of energy expenditure on defense
HA and TH conducted the experiments, analyzed the data, and wrote the manuscript. MQ
performed HPLC and analyzed the derived data. BG and MAK supervised the whole study.
All authors read and approved the manuscript.
We are very grateful to Dr. Bernhard Huchzermeyer for his valuable insights to improve the
discussion. Authors are also thankful to the reviewer for the constructive comments to
improve the manuscript. This study was supported by Pak-US Science and Technology
Cooperation Program co funded by Higher Education Commission, Pakistan and U.S.
Department of State.
839 840 841 842 843 844
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Highlights 1. Antioxidant responses of Desmostachya bipinnata to varying concentrations of NaCl were monitored. 2. The contribution of enzymatic and non-enzymatic antioxidants varied with the applied NaCl treatments. 3. Plants were not able to overcome high salinity induced oxidative stress. 4. The growth of the plants was hampered mainly because of energy expenditure on defense mechanisms.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: