Salt induced modulations in antioxidative defense system of Desmostachya bipinnata

Salt induced modulations in antioxidative defense system of Desmostachya bipinnata

Journal Pre-proof Salt induced modulations in antioxidative defense system of Desmostachya bipinnata Hina Asrar, Tabassum Hussain, Muhammad Qasim, Bre...

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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:

S0981-9428(19)30520-0

DOI:

https://doi.org/10.1016/j.plaphy.2019.12.012

Reference:

PLAPHY 5969

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.

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Salt induced modulations in antioxidative defense system of Desmostachya

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bipinnata

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Hina Asrar1┼, Tabassum Hussain1┼, Muhammad Qasim1, Brent L. Nielsen2

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Bilquees Gul1*, M. Ajmal Khan1

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Institute of Sustainable Halophyte Utilization, University of Karachi, Karachi-75270,

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Pakistan, 2

Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah 84602, USA

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Authors contributed equally to the manuscript.

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*

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Tel.: (9221) 99261032, Fax (9221) 99261340; e-mail: [email protected]

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Declarations of interest: none

Corresponding author:

1

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Abstract

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This study addressed the interactions between salt stress and the antioxidant responses

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of a halophytic grass, Desmostachya bipinnata. Plants were grown in a semi-

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hydroponic system and treated with different NaCl concentrations (0 mM, 100 mM,

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400 mM) for a month. ROS degradation enzyme activities were stimulated by

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addition of NaCl. Synthesis of antioxidant compounds, such as phenols, was

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enhanced in the presence of NaCl leading to accumulation of these compounds under

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moderate salinity. However, when the ROS production rate exceeded the capacity of

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enzyme-controlled degradation, antioxidant compounds were consumed and oxidative

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damage was indicated by significant levels of hydrogen peroxide at high salinity. The

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cellular concentration of salicylic acid increased upon salt stress, but since no direct

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interaction with ROS was detected, a messenger function may be postulated. High

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salinity treatment caused a significant decrease of plant growth parameters, whereas

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treatment with moderate salinity resulted in optimal growth. The activity and

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abundance of superoxide dismutase (SOD) increased with salinity, but the abundance

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of SOD isoforms was differentially affected, depending on the NaCl concentration

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applied. Detoxification of hydrogen peroxide (H2O2) was executed by catalase and

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guaiacol peroxidase at moderate salinity, whereas the enzymes detoxifying H2O2

45

through the ascorbate/glutathione cycle dominated at high salinity. The redox status

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of glutathione was impaired at moderate salinity, whereas the levels of both ascorbate

47

and glutathione significantly decreased only at high salinity. Apparently, the maximal

48

activation of enzyme-controlled ROS degradation was insufficient in comparison to

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the ROS production at high salinity. As a result, ROS-induced damage could not be

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prevented, if the applied stress exceeded a critical value in D. bipinnata plants.

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Keywords: salinity, halophyte, oxidative stress, antioxidative enzymes, non-

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enzymatic antioxidants.

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1. Introduction

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Desmostachya bipinnata, a C4 perennial grass, belongs to the family Poaceae. It has

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high ecological (phytoremediation) and economical (folk medicine and cattle feed)

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potential (Pandey et al., 2013; Shaltout et al., 2016). Its distribution in arid and semi-

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arid regions of the world has drawn researchers’ interest in investigating its salt

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tolerance mechanisms. Such studies will add to our existing understanding and take us

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closer to developing salt tolerant crops with improved survival rates. Fulfilment of

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this long-desired objective has become even more crucial in the context of more land

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becoming saline and the rapidly growing human population.

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Exposure of D. bipinnata to saline conditions affects its photosynthetic performance

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and, therefore, its growth and development. The restriction in CO2 assimilation

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induced at high salinity is associated with an increased dissipation of excitation

65

energy, damage to PSII reaction center components, decline in reactions of the

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Calvin-Benson cycle, and a reduced rate of electron transport (Adnan et al., 2016;

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Asrar et al., 2017). Under such conditions, molecular oxygen serves as an alternate

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sink for photosynthetic electrons. This results in the formation of reactive oxygen

69

species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals (Foyer

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and Shigeoka, 2011).

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The ratio of the electron transport rate to gross photosynthesis increased in D.

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bipinnata at high salinity (Asrar et al., 2017). This implies an increased allocation of

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electrons to processes other than carbon assimilation. We surmise that C4 plants

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suppress photorespiration and provide the photosynthetic electrons with an alternative

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pathway in the form of the Mehler reaction (Bräutigam and Gowik, 2016). In

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addition, the increased energy demands for cellular processes (ion transport, vacuolar

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sequestration, biosynthesis of compatible solutes, etc.) are met with an increased

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activity of the mitochondrial electron transport chain. This accelerates the formation

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of ROS further (Munns and Tester, 2008). Excessive accumulation of ROS can result

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in the execution of cell death. Therefore, strict regulation of their levels is crucial to

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ensure the survival of plants (Mittler et al., 2011).

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Plants are equipped with antioxidant systems to counter the overproduction of ROS

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and avoid or minimize the damage they cause. The enzymatic components includes

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antioxidant enzymes such as superoxide dismutase or SOD (EC 1.15.1.1), ascorbate

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peroxidase or APX (EC 1.11.1.1), catalase or CAT (EC 1.11.1.6), guaiacol peroxidase

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or GPX (EC 1.11.1.7), and glutathione reductase or GR (EC 1.8.1.7). The non-

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enzymatic components, on the other hand, consist of hydrophilic (ascorbate and

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glutathione) and lipophilic (tocopherols and carotenoids) compounds (Foyer and

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Noctor, 2005). Biosynthesis and the activity of the antioxidant system increase under

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stress to stabilize the redox balance (Abogadallah, 2010). Many reports show a

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positive correlation between efficient antioxidants and the salinity tolerance of plants

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(Hamed et al., 2007, 2014; Bouchenak et al., 2012; Benzarti et al., 2014). Exposure to

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moderate or high concentrations of NaCl revealed a significant contribution by

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proteins related to the antioxidative / redox homeostasis in D. bipinnata (Asrar et al.,

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2017). Many proteins were up-regulated or specifically induced to combat a high

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salinity-induced oxidative load. Other proteins increased at moderate salinity, i.e.,

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apparently in the absence of oxidative stress (as indicated by the values for MDA,

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electrolyte leakage and ETR/Ag ratio). The results we obtained highlight the

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importance of an ROS-antioxidant interface to maintain physiological metabolism and

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stimulate acclamatory responses in plants.

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ROS scavenging and its implications in redox homeostasis have been highlighted in

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the past decades (Mullineaux and Baker, 2010; Koyro et al., 2013; Demidchik, 2015).

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However, studies specifying the relative contribution of enzymatic and non-enzymatic

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antioxidants towards salt tolerance are few in number. Therefore, the subject demands

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more research. The components of an antioxidant defense system vary from plant to

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plant (Ksouri et al., 2007; Souid et al., 2016). Further investigation aimed at

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determining the activities of endogenous antioxidants under saline conditions would

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be useful. Chief among the potential benefits would be determining which

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antioxidants should be adopted as markers to develop salt tolerant crops. Such a result

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could have important implications in agro-food biotechnology (Flowers and Muscolo,

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2015).

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A previous study on D. bipinnata revealed that its salt tolerance is based, at least

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partly, on its ability to boost the antioxidative defense response (Adnan et al., 2016).

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Several antioxidants are involved in keeping the ROS below toxic levels. We were

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interested in carrying out an in-depth analysis of the antioxidative defense system to

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search for an answer to the following question. Which components can be used as the

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markers of stress tolerance in this halophyte?

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Therefore, we determined the contribution of various components that were not

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considered in previous studies, such as non-enzymatic antioxidants, antioxidant

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substrates, and SOD isoforms. Additionally, the total antioxidant capacities of plants

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were evaluated to understand the antioxidant system of D. bipinnata.

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Thus, the following questions were specifically investigated:

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1. Is there a correlation between the abundance of ROS scavenging enzymes and

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intensity of salt stress (applied NaCl concentration)? 2. Do all iso-enzymes of SOD respond to salt stress in the same manner?

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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?

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2. Materials and methods

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2.1. Plant material and experimental conditions

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Seeds of D. bipinnata were germinated in a 1:1 mixture of garden soil and manure in

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the growth chamber at 30/20 °C day/night cycle and a photoperiod of 16 h. Three-

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week-old seedlings were transferred to pots (6 ×10 cm; height × diameter) in a wire

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mesh greenhouse and grown under ambient conditions (temperature: 30 ± 2 °C,

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relative humidity: 40 ±10%, PAR: 370 ±50 µmol m−2 s−1). They were watered with

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half-strength basic nutrient solution (Epstein, 1972). After six weeks, the seedlings

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were transferred to pots (18×25 cm; height × diameter) containing Quartz sand. The

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pots were placed in a semi-hydroponic Quick Check System (QCS, Koyro, 2006).

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The ambient conditions were 37 ± 4 °C: 47 ± 12 % RH and 1200 ± 200 µmol m−2 s−1

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PAR.

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After 2 weeks of acclimation, we treated the plants with solutions of various NaCl (0,

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100, and 400 mM) concentrations, termed control, moderate, and high levels of

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salinity, respectively. Preliminary experiments were performed to determine the

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suitable salinity levels. Ten pots (one plant/pot) were used for each salinity treatment

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using a randomized complete block design. The salinity of the nutrient solutions was

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increased gradually by adding 50 mM NaCl per day until the desired concentrations

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were attained. Solutions were changed every 2 weeks to maintain the nutrient levels.

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The duration of the NaCl treatment was 4 weeks.

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2.2.Growth measurement

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Fresh weights (FW) of shoot and roots were measured immediately after harvest. For

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the dry weight (DW) estimation, the shoot and roots were oven-dried at 60 °C for 72

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h and then weighed. Dried plant material was burned in a furnace at 550 °C for 5-7 h

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to obtain ash (inorganic content). The organic content was calculated by subtracting

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ash content from total dry weight.

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Leaf area was calculated with the help of a portable leaf area meter (ADC Bio-

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Scientific Ltd. AM350, England). Specific leaf area (SLA) was calculated as the mean

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leaf area per unit of leaf dry mass. Leaf relative water content (RWC) was determined

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with the procedure reported by Barrs and Weatherley (1962). Leaves (0.5 g) were left

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immersed in distilled water at 4 °C overnight. The leaves were blotted dry and their

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turgid weight (TW) noted. To obtain the dry weight (DW), the leaves were dried for

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48 h at 60 °C. The following formula was used for the RWC calculations:

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RWC (%) = (FW – DW) / (TW – DW) ×100

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The relative decrease in plant biomass (RDPB), relative leaf area ratio (RLAR), and

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salt stress tolerance index (STI) were calculated by using the following equations

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(reviewed by Negrao et al. 2017):

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RDPB = FWC –FWS / FWC ; RLAR = LARS / LARC ; STI = DWS / DWC

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(The subscripts ‘C’ and ‘S’ indicate control and saline treatments, respectively).

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2.3. Determination of H2O2 content

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Hydrogen peroxide (H2O2) content in D. bipinnata leaves was measured according to

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Jessup et al., (1994). Fresh leaf tissue (0.25 g) was homogenized with 5 mL of 3%

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ice-cold trichloroacetic acid (TCA) and centrifuged at 12,000 x g, 4 °C for 15 min.

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Two mL of TCA extract (supernatant) was mixed with 1 mL of 0.5 M potassium

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iodide (KI). The absorbance was recorded at 390 nm (Beckman-Coulter DU-730, UV-

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VIS spectrophotometer). H2O2 concentration was estimated with reference to a

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standard curve for 0-500 µM H2O2.

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2.4. Photosynthetic pigments

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Pigments were extracted from leaf tissue (100 mg) in 80% acetone at 4 °C. Cellular

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debris was removed by centrifugation at 3500 x g for 5 min at 4 °C. The contents of

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pigments were measured by spectrophotometry, according to the equations of

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Lichtenthaler (1987).

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2.5. Determination of ՓPSII and ՓCO2

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The effective photochemical quantum yield of PSII (ՓPSII) and the quantum efficiency

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of CO2 assimilation (ՓCO ) were measured on a matured fully emerged leaf at

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saturating PPFD values for the respective salinity treatments. The ՓPSII was measured

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using a pulse modulated chlorophyll fluorimeter (2500 PAM, Walz, Germany) with

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the following expression as described by Genty et al., (1989):

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ՓPSII = (F'm - Fs)/F'm

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where F'm and Fs are maximal and steady-state fluorescence of light-adapted leaves.

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The ՓCO was measured with a portable photosynthetic system (LICOR-6400,

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Lincoln, NE, USA), according to (Stirling et al., 1991):

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ՓCO = A/PPFD

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where A represents the rate of CO2 assimilation and PPFD is photon flux density on

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the leaf. We express the ratio of ՓPSII and ՓCO as a stress indicator, pointing to a

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discrepancy in the electron transfer photochemistry (Fryer et al., 1998).

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2.6. Antioxidative enzymes activities

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Fresh leaf sample (500 mg) was ground to fine powder with liquid N2 and

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homogenized with 5 mL of extraction buffer (50 mM potassium phosphate buffer, pH

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7.0, 2% (w/v) polyvinylpolypyrrolidone, 1 mM L-ascorbic acid, and 5 mM disodium

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EDTA) in a chilled mortar and pestle. The homogenate was centrifuged at 4 °C for 20

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mins at 12,000 x g. The supernatant was used to determine antioxidant enzymes

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activity of catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase

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(GPX).

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The extraction procedure for superoxide dismutase (SOD) and glutathione reductase

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(GR) was similar to that mentioned above. The only difference was in the pH of

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buffer, i.e., 7.8. Protein concentration was determined, according to Bradford (1976),

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using bovine serum albumin as a standard.

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Catalase (CAT) activity (ξ = 39.1 mM cm-1) was examined according to Aebi (1984).

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The enzyme extract (100 µL) was added to 3 mL of the reaction mixture, containing

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potassium phosphate buffer 50 mM (pH 7.0) and 25 mM H2O2. The decreased

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absorbance due to the disappearance of H2O2 was recorded at 240 nm.

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Ascorbate peroxidase (APX) activity (ξ = 2.8 mM cm-1) was measured by monitoring

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the decrease in absorbance due to the oxidation of ascorbic acid at 290 nm (Nakano

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and Asada, 1981). The reaction mixture consisted of 50 mM potassium phosphate

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buffer (pH 7.0), 0.2 mM EDTA, 0.5 mM ascorbic acid, 2 mM H2O2, and 100 µL

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enzyme extract.

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Activity of guaiacol peroxidase (GPX) (ξ = 26.6 mM cm-1) was calculated according

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to Zaharieva et al., (1999). A reaction mixture containing potassium phosphate buffer

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50 mM (pH 7.0), 2.5 mM H2O2, 2.7 mM guaiacol, and 100 µL enzyme extract was

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prepared. The increase in the absorbance due to formation of tetra-guaiacol was

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measured for 1 min at 270 nm.

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Glutathione reductase (GR) activity (ξ = 6.2 mM cm-1) was measured according to

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Halliwell and Foyer (1978). The enzyme extract (50 µL) was added to a reaction

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mixture containing 100 mM Tris-HCl (pH 7.8), 5.16 mM EDTA, 0.31 mM NADPH,

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and 0.51 mM oxidized glutathione (GSSG). The decrease in absorbance due to

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oxidation of NADPH was recorded at 340 nm and used to calculate the activity.

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The superoxide dismutase (SOD) activity assay was based on the principle of the

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photochemical reduction of nitro blue tetrazolium (NBT), as described by Beyer and

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Fridovich (1987). The enzyme extract (40 µL) was added to a reaction mixture

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containing 0.05 mM NBT, 10 mM L-methionine, 0.22% Triton X-100, and 0.12 mM

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riboflavin in 50 mM potassium phosphate buffer (pH 7.8). One set of test tubes

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containing the reaction mixture was kept under a 40 W fluorescence light. Another set

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was placed in the complete dark for a period of 7 minutes. The increase in absorbance

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at 560 nm due to the formation of formazan under light was measured against the

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control, i.e., the test tube placed in the dark. The absorbance recorded in the absence

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of enzyme extract was taken as 100%. Enzyme activity was calculated as the

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percentage inhibition per min.

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2.7. SOD isozymes

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Native polyacrylamide gel electrophoresis (PAGE) was carried out according to

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Laemmli (1970) on 12% polyacrylamide slab gels, using a Mini-PROTEAN Tetra

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cell (BioRad, Hercules, CA, USA). 40 µg of protein extract was loaded in each gel

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lane. The activity of SOD was visualized by a photochemical NBT reduction method

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(Beauchamp & Fridovich, 1971). Different isoforms of SOD were identified by

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separate incubation of gels in the staining buffer (50 mM potassium phosphate buffer,

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pH 7.8, 0.1 mM EDTA, 28 mM TEMED, 0.003 mM riboflavin and 0.25 mM NBT),

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either with 5 mm H2O2 or 2 mM potassium cyanide (KCN) (Salin and Bridges, 1980).

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KCN and H2O2 inhibit CuZn-SOD activity while H2O2 inhibits that of Fe-SOD. Mn-

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SOD is not inhibited by either KCN or H2O2.

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The gels were immediately scanned with the GenoSens gel documentation system

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(Clinx Science Instruments). Images were analyzed to estimate the relative intensity

251

of each band with CIS 1 D analysis software (Clinx, GenoSens Series, Gel

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documentation system). The intensity of bands from the control treatment was taken

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as a reference and made = 100.

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2.8. Antioxidants substrates and the determination of their redox proportions

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The ascorbate content as reduced (ASC) and total ascorbate [ASC+oxidized ascorbate

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(DHA)] was determined according to Kampfenkel et al. (1995). The reaction mixture

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(4 mL) for measuring the ASC content consisted of leaf TCA extract (6% TCA), 30

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mM potassium phosphate buffer (pH 7.4), 2.5% TCA, 8.4% orthophosphoric acid

259

(H3PO4), 0.8% bipyridyl, and 0.3% ferric chloride (FeCl3). After incubation at 42°C

260

for 40 min in a water bath, the absorbance of the test solution was recorded at 525 nm

261

(Beckman-Coulter DU-730, UV-VIS spectrophotometer).

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For measuring the total ascorbate (ASC+DHA) content, the additional steps included

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the reduction of DHA to ASC by incubation with 0.5 mM dithiothreitol (DTT) at

264

42°C for 15 min, and then the removal of excess DTT with 0.025% N-ethylmaleimide

265

(NEM). The contents of oxidized (DHA) and total ascorbate (ASC+DHA) were

266

estimated with reference curves of dehydroascorbic acid and L-ascorbate solutions,

267

respectively. The concentration of reduced ascorbate (ASC) was calculated by

268

subtracting that of DHA from total ascorbate. The ratio of oxidized and reduced

269

ascorbate was also calculated.

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Glutathione content, as reduced (GSH) and total glutathione [GSH+ oxidized

271

glutathione (GSSG)], was determined according to Anderson (1985) with some

272

modification. The reaction mixture for measuring GSH content consisted of leaf TCA

273

extract (3% TCA), potassium phosphate buffer (pH 7.4) and 50 mM, containing 0.5

274

mM ethylene diamine tetraacetic acid (EDTA) and 0.005% 5,5-dithiobis-(2-

275

nitrobenzoic acid) (DTNB). Absorbance was measured at 412 nm after keeping the

276

reaction mixture at 30°C for 2 min.

277

Total glutathione (GSH+GSSG) content was determined after the reduction of GSSG

278

to GSH by adding 0.2 mM nicotinamide adenine dinucleotide phosphate (NADPH)

279

and 130 mM potassium phosphate buffer (pH 7.4) containing one unit of glutathione

280

reductase (GR). The reaction mixture was incubated at 30°C for 30 min to allow a

281

reaction between the enzyme and the substrate. Absorbance was measured at 412 nm

282

(Beckman-Coulter DU-730, UV-VIS spectrophotometer) and the contents of GSH

283

and GSH+GSSG were estimated with standard curves of glutathione (GSH) solutions

284

as reference. The content of oxidized glutathione (GSSG) was calculated by

285

subtracting GSH from total glutathione. The ratio of reduced to oxidized glutathione

286

was calculated.

287

2.9. Antioxidant capacity assays

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The antioxidant capacity of D. bipinnata was determined using 1,1-Diphenyl-2-

289

picryl-hydrazyl (DPPH; Brand-Williams et al., 1995) and 2,2'-azino-bis3-

290

ethylbenzothiazoline-6-sulphonic acid (ABTS; Re et al., 1999) radical scavenging

291

tests. The reducing potential was estimated with the ferric reducing antioxidant power

292

assay (FRAP; Benzie and Strain, 1996) and total antioxidant capacity using

293

phosphomolybdate complex method (TAC; Prieto et al., 1999).

12

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2.10. Determination of total phenolic (TPC), flavonoid (TFC), proanthocyanidin

295

(PC), and tannin (TTC) contents

296

Total phenolic content (TPC) was determined using the Folin-Ciocalteu colorimetric

297

method (Singleton and Rossi, 1965). Colorimetric methods were also used to quantify

298

total flavonoids (TFC; Chang et al., 2002), total proanthocyanidin (PC; Sun et al.,

299

1998), and total tannins (TTC; Pearson, 1976).

300

2.11. High-performance liquid chromatography (HPLC) analysis

301

Plant samples (0.5 g) were extracted in 40 ml aqueous methanol and 10 ml HCl

302

(Proestos et al., 2006). Extracts were sonicated (15 min) after purging nitrogen (60 s),

303

and then refluxed in a boiling-water bath (120 min). Afterward, the extracts were

304

filtered, methanol was added (up to 100 ml), and filtered again (0.45 µm membrane

305

filter, Millex-HV) before being injected onto a HPLC system.

306

The HPLC system (Shimadzu LC-20AT) included an auto-sampler (SIL-20A), a

307

photo-diode array detector (SPD-M20A), column oven (CTO-20A), Nucleosil C18

308

column (5 µm 100A, 250 x 4.60 mm, Phenomenex), guard column (KJO-4282,

309

Phenomenex), and LC Solution software. Sodium phosphate buffer (50 mM; pH 3.3)

310

and 70% methanol were used as the mobile phase. The volume of the injection was10

311

µL and the flow rate was maintained at 1.2 ml min-1. The gradient program by

312

Sakakibara et al., (2003) was used.

313

2.12. Statistical analyses

314

The statistical software, SPSS, version 24 (SPSS Inc., Chicago, USA) was used for

315

analysis, followed by analysis of variance (ANOVA) to test the significance of all

316

parameters. The Bonferroni test was carried out to determine if significant (p < 0.05)

317

differences existed among means. In addition, a principal component analysis (PCA)

318

was performed with the help of SPSS (v. 24) to correlate the measured parameters on

13

319

plants exposed to salinity treatments. Every principal component was a linear

320

combination of original variables with coefficients equal to eigenvectors of the

321

correlation matrix. Finally, a 2D graphical interpretation of main components was

322

obtained with the same software.

323

3. Results

324

3.1. Growth parameters

325

A significant increase in both root FW (67%) and DW (81%) was recorded at

326

moderate salinity. The shoot biomass (FW and DW) was similar to that of control

327

plants. In contrast, the high salinity treatment caused considerable reduction in FW

328

and DW of shoot and roots. A linear increase with increasing salinity in the organic

329

content of both the shoot and roots of the plants was recorded. A similar trend was

330

observed for the ash (inorganic) content of shoots. However, in roots, it declined

331

substantially at 400 mM NaCl.

332

The RWC was similar (approximately 86%) for all treatments. The maximum value

333

for the specific leaf area was observed at 100 mM NaCl. It decreased by 31% at 400

334

mM NaCl, in comparison to the control treatment plants (Fig. 1). The drastic effects

335

of high salinity on the growth of D. bipinnata were evident from the relative decrease

336

in plant biomass and relative leaf area ratio. The salt tolerance index of these plants

337

was also low (Supplementary Table 1). The plants grown in the presence of 400 mM

338

NaCl showed a lower chlorophyll concentration than those exposed to control and

339

moderate salinity. (Fig. 2).

340

3.2. Oxidative stress indicators

341

A significant increase in the ratio of ՓPSII / ՓCO2 was noted at high salinity. These

342

results were concomitant with a substantial increase in the content of H2O2, which

343

was 33% higher as compared to the control plants. In contrast, in 100 mM NaCl14

344

treated D. bipinnata, the values for ՓPSII / ՓCO2 and H2O2 remained almost constant

345

(Fig. 2).

346

3.3. Antioxidant enzyme activities

347

The 400 mM NaCl-treated D. bipinnata plants showed maximum activities of SOD,

348

GR, and APX, which increased by 35, 35 and 40% respectively, when compared with

349

activities of plants from control treatments. The activity showed the highest

350

correlation coefficient with salinity treatments (r2 = 0.81 for SOD, 0.97 for GR and

351

0.85 for APX) among all the analyzed enzymatic antioxidants. A transient increase in

352

the activity of GPX (209%) and CAT (41%) was recorded at moderate salinity,

353

followed by a decline at high salinity (Fig. 3).

354

3.4. SOD isoforms

355

Fig. 4 shows the changes in the various isoforms of SOD in response to the applied

356

treatments. An analysis of gels revealed an increase in their levels of all SOD

357

isoforms (Mn-SOD, Fe-SOD, and Cu/Zn-SOD) under salinity with the maximum

358

value at 400 mM NaCl. The constitutive expression of Fe-SOD2 was high as

359

compared to that of the other isoforms. The largest change in the expression levels

360

under salinity was recorded for iron-containing SOD isoforms (Fe-SOD1 and Fe-

361

SOD2).

362

3.5. Non-Enzymatic antioxidants: ascorbate and glutathione

363

An investigation of non-enzymatic antioxidants, i.e., ascorbate, and glutathione and

364

their respective pools revealed a linear increase in the oxidized forms. DHA and

365

GSSG levels rose with increasing salinity, but a non-significant change was recorded

366

for reduced forms, i.e., ASC and GSH (Table 1). Total ascorbate content increased

367

(20%) at high salinity when compared to the control. However, total glutathione

368

content showed no significant change. The ASC/DHA ratio was unchanged at 100 15

369

mM NaCl but at 400 mM it decreased significantly. In contrast, a significant decrease

370

in the GSH/GSSG ratio was recorded even at moderate salinity. We noted that total

371

ascorbate showed a high correlation coefficient (r2 = 0.96) with salinity treatments

372

while the accumulation of glutathione seemed unrelated (r2 = 0.25).

373

3.6. Antioxidant capacity – ABTS, DPPH, and FRAP methods

374

The total antioxidant capacity of D. bipinnata leaf extracts did not vary significantly

375

under salinity treatments (r2 = -0.19). However, an assessment of antioxidant activity

376

by DPPH, ABTS, and FRAP methods showed an influence of salinity concentration

377

on these individual tests. Antioxidant activity, based on the capacity of the leaf extract

378

to scavenge DPPH free radicals, was maximum in plants treated with moderate

379

salinity (32% more than that of control plants). It showed the lowest values in the

380

high salinity treatment. Likewise, the highest antioxidant activity evaluated with

381

ABTS test was recorded for plants given the 100 mM NaCl treatment. It remained

382

unchanged at 400 mM NaCl when compared to control-treated plants (Fig. 5).

383

However, the ferric reducing antioxidant potential (FRAP) decreased (approx. 22%)

384

only at high salinity. It presented a highly negative correlation with salinity treatments

385

(r2 = -0.85).

386

3.7. Total phenolic (TPC), flavonoid (TFC), proanthocyanidin (PC), and tannin

387

(TTC) contents

388

The salinity treatments resulted in significant changes in TFC. The highest value was

389

found at moderate salinity and the lowest one at high salinity (Fig. 6). Total

390

polyphenol was increased (28%) under moderate salinity. In contrast, PC did

391

decreased by 41% at high salinity. Total tannin content showed a progressive decrease

392

with increasing NaCl concentration. We noted the highest negative correlation of

393

TTC (r2 = -0.97) and PC (r2 = -0.94) with saline treatments.

16

394

3.8. Phenol profiling

395

The phenolic composition of D. bipinnata leaves was determined using HPLC.

396

Several phenolic compounds were identified, including pyrocatechol, catechin,

397

chlorogenic acid, caffeic acid, salicylic acid, coumaric acid, coumarin, cinnamic acid,

398

quercetin, and kaempferol (Supplementary Fig. 1). In general, most phenolic

399

compounds (pyrocatechol, chlorogenic acid, coumaric acid, coumarin, quercetin, and

400

kaempferol) significantly increased at moderate salinity. However, the high salinity

401

treatment caused a reduction in all but four phenolic acids. Those acids either

402

increased (salicylic and coumaric acids) or remained unchanged (caffeic and cinnamic

403

acids) (Fig. 7). Among all the phenols identified under salinity treatments, the content

404

of catechin (> 2 mg g-1 DW) was the highest while that of quercetin (< 0.15 mg g-1

405

DW) was the lowest. ANOVA revealed a strong positive correlation between

406

moderate salinity and phenolic compounds (except catechin). However, high salinity

407

negatively affected phenolic compounds with the exception of salicylic acid (r2 =

408

0.922) and coumaric acid (r2 = 0.984).

409

3.9. Principal Component Analysis (PCA)

410

The first two principal components (PCs) explained 79.10% of the cumulative

411

variance with PC1 and PC2, contributing 53.7% and 25.4% of the total variance,

412

respectively (Fig. 8). It is evident that the antioxidant enzymes, total and oxidized

413

fractions of ascorbate and glutathione, H2O2 levels, ՓPSII/ՓCO2, and some phenols

414

(coumaric, cinnamic and salicylic acids) were strongly correlated. Therefore, their

415

responses can be separated from the other measured parameters (FW, chlorophyll,

416

antioxidant capacity, ASC/DHA, GSH/GSSG, reduced forms of glutathione and

417

ascorbate, flavonoids, tannins, proanthocyanidin, and most of the phenols) by PC1.

418

However, PC2 revealed a negative correlation of CAT, GPX, and SOD with the other 17

419

enzymatic and non-enzymatic antioxidants. A strong negative correlation was also

420

observed among the antioxidant activity determining methods and GSH, ASC,

421

ASC/DHA, and GSH/GSSG.

422

4. Discussion

423

There are two components of salinity stress: ionic stress and osmotic stress (Munns,

424

2002). As a result, closure of the stomata and an inhibition in gas exchange have been

425

observed due to limited availability of CO2 (Flowers and Colmer, 2015). Especially in

426

the presence of high light intensity, this stressful situation becomes even worse due to

427

increased production of reactive oxygen species (ROS). Their concentration may

428

reach toxic levels (Foyer and Shigeoka, 2011). ROS production has been described as

429

a side reaction of photosynthetic activity. Due to their redox potentials, compounds

430

such as light-activated chlorophyll and reduced ferredoxin can transfer an electron to

431

molecular oxygen to produce an oxygen radical. The probability of this reaction will

432

increase if NADP+, the physiological acceptor of photosynthetic electron transport, is

433

not available. This will be the case if the absorption rate of light quanta significantly

434

exceeds that of NADPH consumption in photosynthesis (Foyer and Noctor, 2005).

435

Under optimal growth conditions, the ROS production rate will be balanced by the

436

rate of their degradation. In many plant species, most of the surplus electrons will be

437

consumed by the glutathione-ascorbate-cycle as described by Asada (Foyer and

438

Shigeoka, 2011). In the case of ROS overproduction, the concerted action of

439

antioxidants and ROS-scavenging enzymes will keep ROS concentrations low. Plant

440

species differ (i) in their contents of antioxidative system and (ii) in their capacity to

441

produce antioxidants under stress. This study addressed the responses of D. bipinnata

442

by comparing the mechanisms involved in oxidative stress tolerance in response to

443

various NaCl treatments.

18

444

4.1. Effects on plant growth

445

In agreement with the previous reports on D. bipinnata (Asrar et al., 2017, 2018) and

446

other halophytic grasses (Flowers and Colmer, 2015), we observed that growth of D.

447

bipinnata was stimulated by addition of 100 mM NaCl to the culture medium.

448

Usually, extensive root growth prevents the accumulation of inorganic ions, mainly

449

Na+, in the shoots (Munns and Tester, 2008). However, we found this was not the

450

case in our study, as we found increased ash content in the shoots. The increased

451

organic content in both shoots and roots under high salinity signifies its contribution

452

in osmotic adjustment (Flowers and Colmer, 2008). Thus, plants were able to

453

maintain their leaf RWC under salinity. The observed reduction in SLA and RLAR at

454

400 mM NaCl indicates the presence of smaller and thicker leaves with fewer

455

stomata, a strategy to conserve water. This may explain the reduction in transpiration

456

(Asrar et al., 2017). In addition, a reduction in RLAR frees more tissue volume to

457

sequester more salts into the vacuoles (Munns, 2002). The damaging effects of toxic

458

salt concentration are apparent in the low values of STI and RDPB (Supplementary

459

Table 1). On the other hand, the relative decrease in plant biomass reflects a switched

460

allocation of available resources from biomass accumulation to energy used in stress-

461

resisting mechanisms (Lavinsky et al., 2015; Nam et al., 2015).

462

4.2. Disturbance in photosynthesis leads to ROS production

463

High values of the Փ PSII/Փ CO2 ratio (Fig. 2) demonstrate the availability of reducing

464

power (NADPH) in excess of its utilization in the Calvin-Benson cycle. This

465

restricted regeneration of NADP+ is known to increase the probability of ROS

466

production (Mehler reaction; Fryer et al., 1998). The increased concentration of H2O2,

467

found in D. bipinnata leaves (Fig. 2) will cause oxidative damage to membrane lipids,

468

proteins, and DNA molecules. It also inactivates enzymes and introduces an

19

469

imbalance in the cellular redox systems (Mittler, 2002; Hamed et al., 2014). The

470

decreased chlorophyll content in response to high salinity treatment (Fig. 2) may be

471

an adaptive strategy to avoid absorption of excessive light. Thereby, limiting ROS

472

production on the expense of photosynthetic capacity.

473

4.3. Components of the antioxidant system and their presumptive functions

474

4.3.1. SOD isoforms

475

The activity of the antioxidant enzyme system (Fig. 3) demonstrates the potential of

476

D. bipinnata to minimize the toxic effects of ROS under salt treatment. SOD, acting

477

as the first line of defense, has been found to increase in several other halophytes

478

(Amor et al., 2005; Lokhande et al., 2011; Bose et al., 2014; Hussain et al., 2015). We

479

found this increased activity concomitant with the up-regulation of SOD isoforms

480

(Figure 4). Among these, the largest increase in the content of Fe-SODs suggests they

481

play a predominant role in scavenging chloroplastic-O2.- (Myouga et al., 2008). The

482

increase may also be explained as an adaptive strategy of plants to bind excessive Fe -

483

- a redox active metal ion with the potential to generate the most damaging ROS (i.e.

484

OH· ) -- from participation in Fenton reaction. This explanation is intriguing, as a

485

reduction in the expression of a vacuolar iron-sequestering protein was reported

486

previously (Asrar et al., 2018). In addition, increased expression of other SOD

487

isoforms (i.e. Mn-SOD, Cu/Zn-SOD) has been observed. They are found in the

488

mitochondria, cytosol, and apoplastic regions. This suggests a strategic control of the

489

plant’s defense system in detoxifying ROS at their places of origin.

490

Of particular importance is the accumulation of Cu/Zn-SODII, which resides in close

491

proximity to photosystem I (PSI). Therefore, SOD may actively scavenge ROS

492

generated in this region (Kliebenstein et al., 1998). This is quite interesting, as our

493

previous work proposed salinity-induced damage in the PSI electron transport chain 20

494

(Asrar et al., 2017). However, the results we obtained in this study are in contrast to

495

those reported for some salt sensitive plants.

496

In pea plants, an increased expression of one SOD isoform is accompanied by

497

inhibited expression of other isoforms (Hernandez et al., 1995; Gomez et al., 1999).

498

This may characterize an important difference of the salt tolerance mechanism of C3

499

plants (such as pea) and our test species, which is a C4 plant. Apparently it is

500

important for this plant’s type of C4 pathway to efficiently scavenge ROS produced in

501

bundle sheath chloroplasts as well as in mitochondria.

502

Summarily, the differential accumulation of the various isoforms of enzymatic

503

antioxidant SOD enhances the tolerance of D. bipinnata to oxidative stress by

504

catalyzing the conversion of O2.- into H2O2 at various sub-cellular sites. However, the

505

contribution of other related antioxidant enzymes and their isoforms in combating

506

oxidative stress cannot be neglected and should be examined in future studies. These

507

findings also answer one of the queries of this study: all isozymes of SOD do not

508

respond to salt stress in the same manner.

509

4.3.2. Antioxidant enzymes

510

The enhanced SOD activity led to increased H2O2 content causing secondary

511

oxidative stress in our test species. Similar observations have been reported for other

512

halophytes as well (Amor et al., 2005; Lu et al., 2016). Enzymes such as CAT, APX,

513

GPX, and GR, among others, regulate the levels of H2O2. The considerably high

514

activities of APX and GR indicated the involvement of the ASC/GSH cycle to

515

scavenge H2O2 under high salinity treatment. On the other hand, CAT and GPX were

516

involved in the detoxification of H2O2 mainly at moderate salinity. This observed

517

correlation between applied NaCl concentrations and the abundance of ROS-

518

scavenging enzymes answers our first question in this study. As a response to salt

21

519

stress, the redox state of the glutathione pool became more oxidized (Table 1).

520

Increased concentrations of GSSG will enhance the regeneration of NADP+. The

521

competition for electrons will reduce ROS formation, as discussed by Foyer and

522

Noctor (2011).

523

4.3.3. The ascorbate-glutathione cycle

524

In the presence of moderate salinity, we observed in our test species, an unchanged

525

ASC/DHA ratio (Table 1). Previously, a low value for MDA and electrolyte leakage

526

was recorded in response to similar treatment (Asrar et al., 2017, 2018). Apparently,

527

ROS production induced by moderate NaCl concentrations can be balanced by the

528

Asada pathway. Cellular ROS concentration is kept low and therefore damage, such

529

as peroxidation of membrane lipids, is prevented. From this conclusion, it may be

530

reasoned that salinity tolerance of D. bipinnata is limited by the capacity of the

531

ascorbate-glutathione cycle to compensate for ROS over-production and, thus, control

532

cellular ROS concentration.

533

Stress induced over-expression of respective genes results in an increased abundance

534

of the enzymes controlling the above mentioned reactions. While such enzyme

535

production takes time, ROS detoxification by direct reaction with antioxidants is an

536

immediate reaction, saving other cell components from peroxidation. For a better

537

understanding of this part of the ROS defense system, we have used four different

538

assays, DPPH, ABTS, TAC, and FRAP (Fig. 5). As described in the literature (Melo

539

et al., 2008; Rodrigues et al., 2011; Berłowski et al., 2013), these tests allow

540

evaluation of the capacity of a cell extract to detoxify ROS, and measurements of

541

individual concentrations of some major antioxidants. Our results show that treatment

542

of D. bipinnata plants with moderate salinity results in the stimulation of antioxidant

543

capacity and is associated with enhanced cellular concentration of phenols and

22

544

flavonoids (Fig. 6). Antioxidants do not undergo a regeneration cycle. Thus, their

545

concentrations were significantly reduced in the presence of high NaCl stress, when

546

the ROS production rate exceeded the detoxification capacity of enzyme-controlled

547

reactions such as the Asada cycle. Results of the correlation analysis indicate that

548

cellular antioxidative capacity is linked to the concentrations of phenols and

549

flavonoids. On the other hand, only a low correlation was found with respect to

550

concentrations of other compounds, such as tannins and pro-anthocyanidin. Thus, we

551

conclude that the ABTS assay may be a key tool to rank the antioxidative defense

552

response in related plant species.

553

4.3.4. Phenols

554

Phenols have been shown to improve tolerance to other types of stresses as well, such

555

as heavy metal stress (Bravo, 1998; de Groot and Rauen, 1998; Simiæ et al., 2007;

556

Maurya and Devasagayam, 2010). Therefore, we applied HPLC analysis to evaluate

557

the effects of NaCl on the abundance of several phenolic compounds (Fig. 7).

558

Increased contents of kaempferol and quercetin have to be interpreted in the context

559

of their involvement in the biosynthesis of glutathione (Moskaug et al., 2005).

560

Kampeferol also plays an important role in control of meristematic activities, being a

561

cofactor of auxin (Janesen, 2002).

562

Salicylic acid is known to have two functions: it can act as an antioxidant (Simić et

563

al., 2007) as well as a second messenger signaling ROS-caused stress events

564

(Pirasteh-Anosheh and Emam, 2018; Kim et al., 2018). While the concentration of

565

most antioxidants decreased in the presence of 400mM NaCl, salicylic acid

566

concentration remained at an increased level. In a similar way, the cellular

567

concentration of coumaric acid was stimulated by addition of NaCl and did not

568

correlate with the antioxidative capacity of cells. We, therefore, conclude that in D.

23

569

bipinnata these two compounds are not involved in ROS detoxification, as found by

570

Rezazadeh et al., (2012) e. Rather, their role as a pro-oxidant can be presumed. These

571

analyses allowed us to answer another question: all phenolic compounds did not

572

respond in the same way to changes in the degree of NaCl stress.

573

5. Conclusion

574

The present study provides information about responses to moderate and high salinity

575

of the antioxidative defense system of D. bipinnata. We have analyzed plant samples

576

after an extended period of salt treatment. We have observed different concentrations

577

of antioxidant compounds and enzymes involved in ROS degradation. Our

578

interpretation of these observations is summarized in fig. 9.

579

Our results differ from earlier observations of other research groups (Hernandez et al.,

580

1995; Gomez et al., 1999). This can because we have used D. bipinnata as an

581

experimental plant, which employs C4 metabolism for assimilation of CO2, while in

582

the cited publications stress responses of a C3 plant were analyzed.

583

A more detailed analysis of biochemical pathways resulting in the observed

584

differences requires identification of the type of C4 photosynthesis used by D.

585

bipinnata. From earlier experiments, we could conclude that D. bipinnata is using

586

PEP carboxykinase type CO2 fixation. However, in accordance with findings of

587

Schlüter et al. (2016), we speculate that a modification of equilibria between

588

metabolites can be achieved under stress by tuning activities of metabolic pathways.

589

Therefore, we currently are unable to describe plant responses to NaCl at the

590

biochemical level in detail, as we lack information such as measurement of stress

591

effects on the genetic control of expression of enzymes of the C4 pathway.

592

In this context, stress-induced changes in the patterns of SOD isoenzymes indicate

593

that sites of ROS production vary in activity, depending on the degree of salt stress.

24

594

As ROS production is an indicator of bottlenecks in metabolism, it may be concluded

595

that in the presence of 100 mM NaCl and 400mM NaCl different intermediates will

596

build up. These intermediates may be substrates of different secondary metabolic

597

pathways. Therefore, we have found different patterns of secondary metabolites in the

598

presence of different NaCl concentrations.

599

HPLC analysis of plant extracts showed that salt treatment resulted in increased

600

cellular salicylic acid concentration. It may be postulated that this acts as a signal that

601

will stimulate a transcription factor controlling the expression of NaCl-responsive

602

genes. Thus, salicylic acid may be used as an indicator of NaCl stress, but apparently

603

it does not tell the degree of stress.

604

Recognizing moderate stress is possible if we measure concentrations of phenolic

605

antioxidants consumed by ROS detoxification and present as a proof of concept. If

606

stress exceeds a threshold value, concentrations of these antioxidants are too low to

607

allow a reliable ranking of stress degree. In our experiments, this is the case in the

608

presence of 400 mM NaCl. Our results also allow us to state that the concentration of

609

antioxidants and the activity of ROS degrading enzymes was sufficient to save the

610

plant from ROS stress at moderate salinity treatment. These conclusions are

611

summarized in Fig. 9. As we do not know the exact pathway of C4 photosynthesis in

612

D. bipinnata, we have shown only one hypothetical cell containing one chloroplast

613

and one mitochondrion instead of the two cell types involved.

25

614

Figures:

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

622 623

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

629

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

636

01 4

C

+

+

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

29

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).

707 708

32

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

DHA -1

(µmol g FW)

Total Ascorbate -1

(µmol g FW)

GSH (nmol g-1 FW)

ASC/DHA

GSSG Total Glutathione (nmol g-1 FW) (nmol g-1 FW)

GSH/GSSG

0

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

100

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

400

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

33

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).

34

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.).

35

786

Supplementary Data:

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.

36

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)

RDPB

RLAR

100

-0.09 ± 0.03a

1.01 ± 0.03a

400

-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

820 821

D MANUSCRIPT

822

1. Antioxidant responses of Desmostachya bipinnata to varying concentrations of NaCl were

823

monitored.

824

2. The contribution of enzymatic and non-enzymatic antioxidants varied with the applied

825

NaCl treatments.

826

3. Plants were not able to overcome high salinity induced oxidative stress.

827

4. The growth of the plants was hampered mainly because of energy expenditure on defense

828

mechanisms.

829

Authors’ Contribution:

830

HA and TH conducted the experiments, analyzed the data, and wrote the manuscript. MQ

831

performed HPLC and analyzed the derived data. BG and MAK supervised the whole study.

832

All authors read and approved the manuscript.

833

Acknowledgement:

834

We are very grateful to Dr. Bernhard Huchzermeyer for his valuable insights to improve the

835

discussion. Authors are also thankful to the reviewer for the constructive comments to

836

improve the manuscript. This study was supported by Pak-US Science and Technology

837

Cooperation Program co funded by Higher Education Commission, Pakistan and U.S.

838

Department of State.

Highlights

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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: