Environmental and Experimental Botany 128 (2016) 79–90

Differential fine-regulation of enzyme driven ROS detoxificationnetwork imparts salt tolerance in contrasting peanut genotypes

Koushik Chakraborty*, Sujit K. Bishi, Nisha Goswami, Amrit L. Singh, Pratap V. ZalaICAR-Directorate of Groundnut Research, Junagadh 362001, Gujarat, India

A R T I C L E I N F O

Article history:Received 24 February 2016Received in revised form 2 May 2016Accepted 2 May 2016Available online 3 May 2016

Keywords:Antioxidant defenseGroundnutOxidative stressSalt stressSuperoxide radical

A B S T R A C T

The present study was aimed to identify the major ROS detoxification pathway in peanut under salinitystress. Pot experiment was conducted with six peanut genotypes (differing in salt-sensitivity) and fourlevels of salt stress. Higher level of salt stress led to severe plant mortality and reduction in membranestability especially in sensitive genotypes. Higher ROS accumulation in sensitive genotypes (NRCG 357and TMV 2) as compared to the tolerant ones (Somnath, TPG 41, CS 240) was confirmed by bothspectrometry and in situ histo-chemical staining. Salinity stress changed the cellular antioxidant pool,where the levels of total ascorbate and proline increased in all the genotypes, but the total glutathionecontent showed significant reduction with more pronounced effect in sensitive genotypes. Major changesin POD and CAT activities was observed in response to salt stress, indicating POD as the major H2O2

detoxifying enzyme in tolerant genotypes. The POD activity was supplemented by CAT activity insensitive genotypes, where there was relatively higher ROS load. The SOD showed minimal up-regulationunder salt stress with undistinguishable difference between tolerant and sensitive genotypes, while APXand GR showed almost no induction, suggesting nominal association of these enzymes with overall salttolerance in peanut.

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1. Introduction

Salinity is one of the major abiotic stresses limiting plantgrowth and productivity as nearly 7% of the total territorial areaand 20% of the irrigated arable land is affected by soil salinityglobally (Parihar et al., 2015). Peanut (Arachis hypogaea L.), animportant legume, preferred both as oilseed and confectionarypurposes globally, has been reported to be moderately saltsensitive and shows restriction in growth and yield after crossingthe threshold level of soil salinity (Singh et al., 2008). As peanut iscultivated mostly in marginal and resource poor soil, so developingsalinity tolerant genotypes would definitely help to expand its areaof cultivation in non-traditional saline soils. Although, consider-able efforts were made in the past for developing salt-tolerantpeanut cultivars, but very limited success was tasted mostly due to

Abbreviations: APX, ascorbate peroxidase; CAT, catalase; GR, glutathionereductase; GSSG, oxidized glutathione; GSH, reduced glutathione; GPX, glutathioneperoxidase; MSI, membrane stability index; POD, peroxidase; ROS, reactive oxygenspecies; SOD, superoxide dismutase; SOR, superoxide radical; TBARS, thiobarbituricacid reactive substances.* Corresponding author at: Plant Physiology Department, ICAR-Directorate of

Groundnut Research, Junagadh 362001, Gujarat, India.E-mail address: koushikiari@gmail.com (K. Chakraborty).

http://dx.doi.org/10.1016/j.envexpbot.2016.05.0010098-8472/ã 2016 Elsevier B.V. All rights reserved.

lack of understanding of key mechanisms of salt tolerance in thiscrop. As we know, salt tolerance is a complex trait consisting ofvarious mechanisms; hence detailed understanding of each of thecomponents is absolutely essential for developing salt tolerantgenotypes (Flowers, 2004).

Even under normal environmental conditions plants tend toproduce reactive oxygen species (ROS) essentially as byproducts ofphotosynthesis, respiration and photorespiration (Apel and Hirt,2004; Mittler, 2002). Environmental stresses including salinityfurther aggravate the production of ROS, which causes severeoxidative damage to the plants growing in saline environment(Gupta and Huang, 2014). Salt stress can affect plant growth andmetabolism due to both osmotic (dehydration) and ionic (Na+ andCl� toxicity) effects (Flowers, 2004). Due to the lowering of waterstatus, plants tend to close their stomata partially in order toprevent higher water loss through transpiration under salt stress,resulting in reduced CO2 supply to leaves and unfavourable CO2/O2

ratio in chloroplasts (Remorini et al., 2009). Such deprivation ofinternal CO2 concentration and increased rate of photorespirationinduces the oxygenase activity of Rubisco resulting in toxicsuperoxide radical formation (Hsu and Kao, 2003) and higher H2O2

production in the leaf tissue (Hernandez et al., 2000). The mostcommonly occurring ROS in plants includes hydrogen peroxide

80 K. Chakraborty et al. / Environmental and Experimental Botany 128 (2016) 79–90

(H2O2), superoxide anions (O�2), hydroxyl radicals (�OH) andsinglet oxygen (1O2), which interferes with normal cellularmetabolism by oxidizing proteins, lipids and DNA and othercellular macromolecules under salt stress (Ahmad et al., 2016).

To counterbalance these, plants have well-defined network ofROS detoxification system having an array of enzymes and lowmolecular weightorganicmetaboliteseitherworkingindependentlyor in combination. Superoxide in chloroplast is dismutated bysuperoxide dismutase (SOD) into H2O2, which is decomposed by avariety of peroxidases such as ascorbate peroxidase (APX), glutathi-one peroxidase (GPX) and phenol peroxidase (Asada, 1999) intowater by using various reducing agents. On the other hand, H2O2

produced in the peroxisome as a result of photorespiration isdecomposed primarily by catalase (CAT) (Dat et al., 2000). Bothperoxidases (APX and POD) and CAT decompose H2O2, but in generalperoxidases have much higher affinity to H2O2 than CAT (Mittler,2002; Abogadallah, 2010), indicating the fact that peroxidases couldbe associated with ROS scavenging at lower cellular H2O2

concentration, while CAT removes H2O2 at much higher concentra-tion. Under salt stress, higher ROS accumulation could cause severecell damages, however, lower levels of ROS particularly H2O2 issuggested to work as secondary signals that activate the stresstolerance mechanisms (Desikan et al., 2001).

Different plant species respond differentially by modulating theirantioxidative machinery. For instance, salinity stress in cotton,increased the activities of SOD, peroxidase (POD), glutathionereductase (GR) but decreased the activities of CAT and APX (Gossettet al., 1994); where as in rice, strong up-regulation of SOD, APX andGPX activities were observed in response to salt stress with littlechange in GR and decline in CAT activities (Lee et al., 2001). Higherinduction of SOD, APX and POD was observed in tomato withincreasing salinity levels (Mittova et al., 2004). Abogadallah et al.(2010) reported crucial role of SOD, POD, APX and GR in salt tolerancemechanismofC4barnyardgrass,whileCATactivityalthough inducedunder salt stress, but had negligible association with salt tolerance.Under salt stress, the antioxidant system of the halophyte, Limoniumsinense, is activated by up-regulation of CAT, SOD and POD andeffectively scavenges reactive oxygen species in order to maintaingrowth under high external Na+ concentration (Zhang et al., 2014).

From the existing evidences, it seems that plants do differ inexecuting the antioxidant defense strategy at the mechanistic levelunder different abiotic stresses. Not only that, even under saltstress every species does not necessarily require to up-regulate thefull set of antioxidant enzymes for achieving salt tolerance. Ratherone or few specific component of the whole defense system playscrucial role, which may well vary from one crop to another. Thus, itis very much pertinent to identify crop specific components of ROSdetoxification pathway, if any, that are involved in imparting salttolerance in peanut. To the best of our knowledge, specificinformation on salt tolerance mechanism of peanut in terms of fineregulation of ROS detoxification pathway has not been well workedout till date. Such intricate knowledge will definitely help inunderstanding salinity tolerance mechanism in peanut and internwould benefit in developing salt tolerant genotypes. Hence, thepresent study was aimed to know (i) how the specific component(s) of antioxidant defense system imparts salinity tolerance inpeanut through ROS detoxification? and (ii) is the role of specificcomponent(s) differing between sensitive and tolerant peanutgenotypes?

2. Materials and methods

2.1. Experimental condition and plant material

For the present study, a pot experiment was conducted insummer (dry season) 2015 at ICAR-Directorate of Groundnut

Research, Junagadh, India. Based on our initial field/lab screening(data not shown), six genotypes viz. ‘NRCG 357’, ‘CS 240’, ‘TMV 2’,‘Girnar 1’, ‘TPG 41’ and ‘Somnath’ were selected in the presentstudy, having enough diversity amongst themselves to accommo-date the maximum representation of cultivated peanut genotypes(see Supplementary Table S1 for individual genotype character).

The plants were subjected to four different levels of salttreatment (0, 25, 50 & 100 mM NaCl) through irrigation waterstarting from 2 weeks after sowing (after early establishment ofthe seedlings). The plants were watered every alternate day tomaintain soil moisture level at field capacity. The wholeexperiment was conducted in two-factor completely randomizeddesign with 10 replicates (in the form of pots) per treatmentcombination, where 5 plants were kept per pot to maintain anuniform plant stand throughout the experimental period. Pro-longed salt treatment for six weeks resulted in significantdevelopment of soil salinity leading to severe plant mortality insensitive genotypes under 100 mM NaCl treatment. As we lostalmost all the plants under 100 mM NaCl treatment, the finalexperiment was restricted to only three levels of salt stress (0, 25 &50 mM).

All the studied parameters under present experiment wereinvestigated at 60 days after sowing (about 6 weeks of salttreatment). For this, uniform samples were collected from thirdfully matured leaf on the main axis randomly for all thephysiological and biochemical estimations and total RNA extrac-tion for gene expression studies.

2.2. Estimation of soil parameters and membrane stability index

To study the level of stress developed during the experimentalperiod, soil samples were collected at 60 days after sowing (DAS),oven dried and finely ground. For estimation of Na+ content, theextraction was done in neutral 1 N ammonium acetate solution(Hanway and Heidel, 1952) and the pH and electrical conductivityof the soil samples were measured using portable Hanna-makepH-EC meter in saturation extract with distilled water at a ratio of1:2.5.

Membrane stability index (MSI) was estimated by measuringthe electrical conductivity of leaf samples (100 mg) in 10 mL doubledistilled water by heating at 40 �C for 30 min and 100 �C for 10 minas described by Chakraborty et al. (2012).

2.3. Determination of superoxide radical, H2O2 content and lipidperoxidation

To determine the level of oxidative stress, different keycomponents like superoxide and hydrogen peroxide content andlipid peroxidation level was measured. Superoxide radical contentwas estimated by its capacity to reduce nitroblue tetrazoliumchloride (NBT) and the absorption of end product was measured at540 nm (Chaitanya and Naithani, 1994). Hydrogen peroxide wasestimated by forming titanium-hydro peroxide complex (Rao et al.,1997). One gram leaf was ground with liquid nitrogen and the finepowder was mixed with 10 mL cooled acetone in a cold room. Thefiltered mixture was added with 4 mL titanium reagent and 5 mLammonium solution to precipitate the titanium-hydro peroxidecomplex which was further dissolved 10 mL of 2 M H2SO4 andabsorbance was recorded at 415 nm against blank.

The level of lipid peroxidation was measured in terms ofthiobarbituric acid reactive substances (TBARS) content (Heathand Packer, 1968). Leaf sample (0.5 g) was homogenized in 10 mL0.1% trichloro-acetic acid (TCA) and centrifuged at 15000g for15 min. One mL of supernatant was mixed with 4 mL of 0.5%thiobarbituric acid (TBA) in 20% TCA and heated at 95 �C for 30 minfollowed by cooling in ice bath. After centrifugation at 10,000g for

K. Chakraborty et al. / Environmental and Experimental Botany 128 (2016) 79–90 81

10 min, the absorbance of the supernatant was recorded at 532 nm.The TBARS content was calculated according to its extinctioncoefficient e = 155 m M�1 cm�1. The values for non-specific absor-bance at 600 nm were subtracted.

2.4. In situ localization of hydrogen peroxide (H2O2) and superoxideradical (O2

�)

In situ localization of hydrogen peroxide (H2O2) and superoxideradical (O2

�) were examined based on histochemical staining by3,3-diaminobenzidin (DAB) and nitroblue tetrazolium (NBT)respectively. DAB (1 mg mL�1) was dissolved in distilled waterand adjusted to pH 3.8 with KOH (prepared freshly in order to avoidauto-oxidation) (Ramel et al., 2009). NBT (1 mg mL�1) wasdissolved in 10 mM potassium phosphate buffer (pH 7.8) contain-ing 10 mM NaN3 (Ramel et al., 2009). Leaflets of 60 DAS plants wereimmersed and incubated for 12 h in DAB and NBT solutionsseparately in dark at room temperature. Stained leaflets werebleached twice in acetic acid: glycerol: ethanol (1:1:3) (v/v/v)solution at 80 �C for 5 min. Leaflets were then stored in a glycerol:ethanol (1:4) (v/v) solution until photographed. H2O2 wasvisualized as a brown color due to DAB polymerization whileO2

�was visualized as a blue color of NBT precipitation produced onbleached leaflets.

2.5. Antioxidant enzyme assay

The extract for all the antioxidant enzymes (SOD, APX, GR, PODand CAT) was prepared as described earlier (Chakraborty et al.,2015). The extract was prepared by freezing 1 g of leaf samples inliquid nitrogen followed by grinding in 10 mL extraction buffer(0.1 M phosphate buffer, pH 7.5, containing 0.5 mM EDTA in case ofSOD, GR, POD, CAT and 1 mM ascorbic acid in case of APX). Afterfiltering the extract was centrifuged for 20 min at 15000g and thesupernatant was used as enzyme.

Total SOD (EC 1.15.1.1) activity was estimated by the inhibitionof the photochemical reduction of nitroblue tetrazolium (NBT) bythe enzyme (Dhindsa et al., 1981). The reaction was started byadding 2 mM riboflavin (0.1 mL) in 3 mL of reaction mixture(13.33 mM methionine, 75 mM NBT, 0.1 mM EDTA, 50 mM phos-phate buffer (pH 7.8), 50 mM sodium carbonate, 0.1 mL enzymeextract) and placing the tubes under two 15 W fluorescent lampsfor 15 min. The absorbance was recorded at 560 nm, and one unit ofenzyme activity was taken as that amount of enzyme, whichreduced the absorbance reading to 50% in comparison with tubeslacking enzyme. Catalase (EC 1.11.1.6) was assayed by measuringthe disappearance of H2O2 (Aebi,1984) in a reaction mixture (3 mL)consisting of 0.5 mL of 75 mM H2O2 and 1.5 mL of 0.1 M phosphatebuffer (pH 7) and 50 mL of diluted enzyme extract. The decrease inabsorbance at 240 nm was observed for 1 min and enzyme activitywas computed by calculating the amount of H2O2 decomposed.Peroxidase (EC 1.11.1.7) activity was measured in terms of increasein absorbance due to the formation of tetra-guaiacol at 470 nm andthe enzyme activity was calculated as per extinction coefficient ofits oxidation product, tetra-guaiacol e = 26.6 m M�1 cm�1 (Castilloet al., 1984). The reaction mixture contained 50 mM phosphatebuffer (pH 6.1), 16 mM guaiacol, 2 mM H2O2 and 0.1 mL enzymeextract. The mixture was diluted with distilled water to make upfinal volume of 3.0 mL. Enzyme activity is expressed as mmol tetra-guaiacol formed per min per mg protein.

Ascorbate peroxidase (EC 1.11.1.11) was assayed by recording thedecrease in optical density due to ascorbic acid at 290 nm (Nakanoand Asada, 1981). The 3 mL reaction mixture contained 50 mMpotassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.1 mMEDTA, 0.1 mM H2O2, 0.1 mL enzyme and water to make a finalvolume of 3.0 mL in which 0.1 mL of H2O2 was added to initiate the

reaction. Decrease in absorbance was measured spectrophoto-metrically and the activity was expressed by calculating thedecrease in ascorbic acid content using standard curve drawn withknown concentrations of ascorbic acid. Glutathione reductase (EC1.8.1.7) was assayed as per the method of Smith et al. (1988). Thereaction mixture containing 66.67 mM potassium phosphatebuffer (pH 7.5) and 0.33 mM EDTA, 0.5 mM DTNB in 0.01 Mpotassium phosphate buffer (pH 7.5), 66.67 mM NADPH,666.67 mM GSSG and 0.1 mL enzyme extract. Reaction was startedby adding 0.1 mL of 20 mM GSSG in 3 mL reaction mixture and theincrease in absorbance at 412 nm was recorded spectrophotomet-rically and the activity was expressed as mmol of oxidizedglutathione reduced per mg protein per min.

2.6. Estimation of non-enzymatic molecular antioxidant

Free proline content in the leaves was determined following themethod of Bates et al. (1973). Leaf samples (0.5 g) werehomogenized in 5 mL of sulfo-salicylic acid (3%) and 2 mL ofextract was mixed with 2 mL of glacial acetic acid and 2 mL ofninhydrin reagent boiled in water bath at 100 �C for 30 min. Aftercooling 6 mL of toluene was added to it and the chromophorecontaining toluene was separated and absorbance was read at520 nm. For carotenoid estimation 50 mg of leaf material wasplaced in 10 mL of DMSO and incubated in 65 �C for 4 h (Hiscox andIsraelstam, 1979). After cooling to room temperature the absorp-tion of this organic solvent were recorded at 470 nm against DMSOblank and the pigment concentration was calculated as describedby Lichtenthaler and Wellburn (1983).

Total ascorbate content and its reduced (AsA) and oxidized(DHA) form was estimated by dipyridyl-ferric chloride reagentsmethod as described by Gossett et al. (1994). For this, fresh leaftissue (1 g) was homogenized in cold 5% metaphosphoric acid andafter centrifugation at 22,000g, the supernatant was used for bothAsA and DHA estimation. Total ascorbate was estimated afterreduction of DHA to AsA by DTT, and the concentration of DHA wasdetermined from the difference between the total ascorbate andAsA. Separate reaction mixture was prepared for total ascorbate(0.3 mL plant extract; 0.75 mL of 150 mM phosphate buffer, pH 7.4containing 5 mM EDTA; 10 mM DTT) and AsA (0.3 mL plant extract;0.75 mL of 150 mM phosphate buffer, pH 7.4 containing 5 mMEDTA; 0.3 mL water) and incubated for 10 min at RT. To removeexcess DTT 0.15 mL of N-ethylmaleimide (0.5%) was added in all thereaction mixtures of total ascorbate. Both the sets of reactionmixtures were added with TCA (10%), ortho-phosphoric acid (44%),dipyridyl (4% in 70% ethanol) and FeCl3 (0.3%) and incubated for40 min at 40 �C for colour development, which was subsequentlyread at 525 nm and concentration of all the fractions of ascorbatepool were worked out using standard solutions of L-ascorbate.

Both the oxidized (GSSG) and reduced (GSH) glutathione wereassayed as per the method of Smith (1985). The extract wasprepared by homogenizing 1 g fresh leaf material in 10 mL of cold5% metaphosphoric acid and centrifuged at 22000g for 15 min at4 �C. For total glutathione (GSH + GSSG) 1 mL of supernatant isneutralized with 1.5 mL of 0.5 M phosphate buffer (pH 7.5),followed by addition of 50 mL of water and for GSSG assay another1 mL of supernatant is neutralized with 1.5 mL of 0.5 M phosphatebuffer (pH 7.5), followed by addition of 50 mL of 2-vinylpyridine, tomask the GSH, and the contents of the tube are vortexed until anemulsion formed. The tube is then incubated for 60 min at roomtemperature. The reaction is started by adding 0.1 mL of sampleextract in 3 mL of reaction mixture consisting of 0.2 mM NADPH,100 mM of phosphate buffer (pH 7.5), 5 mM EDTA, 0.6 mM DTNBand 3 units of purified GR enzyme (Sigma-Aldrich; CAS Number9001-48-3). The reaction rate was monitored by measuring thechange in absorbance at 412 nm for 1 min and concentration of

82 K. Chakraborty et al. / Environmental and Experimental Botany 128 (2016) 79–90

glutathione was quantified by referring to a standard curve basedon GSH in the range of 0–50 mM mL�1.

2.7. Gene expression study

Total RNA from leaf tissue of control and stressed plants wereisolated using RNAEasy Kit (Qiagen) according to manufacturer’sprotocol with minor modifications. To ensure least contaminationof genomic DNA, the total RNA extract was subjected to DNase Itreatment (using Qiagen kit as per manufacturer’s protocol).Further the integrity of the RNA was confirmed in the gel andabsorbance of the isolated RNA was recorded by NanodropSpectrophotometer (ND 1000). Approximately 2 mg of total RNAwas used for cDNA synthesis using First strand cDNA synthesis kit(Thermo SCIENTIFIC) and the synthesized cDNA was confirmed byPCR using all cDNA samples (about 100 ng) as template usingb-actin as primer. To test gene specific primers (see Supplemen-tary Table S2 for sequence details), the above experiment wascarried out for each set of primers. Except for CAT, primers for allother genes showed expected sizes of amplicon after 30 cyclereaction at 60 �C annealing temperature.

Changes in transcript expression of the above genes werestudied by Real-Time quantitative PCR using QuantiFast SYBRGreen PCR reaction kit (Qiagen,USA). The reaction mixturecontained about 100 ng of cDNA, 0.16 mM of primers and 12.5 mLof QuantiFast SYBR Green PCR mix. The volume of reaction wasmaintained to 25 mL by sterile nuclease free water. Reactions wererun in StepOnePlusTM Real-Time PCR System (Applied Biosystem)and conditions were set as: 95 �C–5 min for 1 cycle; 95 �C–10 s and60 �C–30 s for 40 cycles. At the end of the PCR cycles, a melt curveanalysis was carried out to determine the specificity of amplifica-tion. The fold changes in relative transcript abundance in stressedplants were compared to control plants by comparative 2�DDCt

method (Schmittgen and Livak 2008). The Ah-actin gene was usedas internal control to normalize the PCR reactions.

2.8. Statistical analyses

All the data recorded were the mean values � standard error(mean) of 5–6 independent biological replicates. The experimentwas conducted in two-factor completely randomized design andthe data were subjected to two-way ANOVA as per theexperimental design. The least significant differences (LSDP=0.05)for NaCl � Genotypes interaction were considered statisticallysignificant while comparing different treatment combinations(Gomez and Gomez, 1984).

3. Results

3.1. Effect of salinity treatment on soil parameters and plant mortality

Imposition of salinity treatment for prolonged period of time (6weeks) resulted in significant build-up of soil salinity at the time ofsampling in the present study. Both soil ECe value and Na+ contentincreased significantly with the increase in salt stress level, whilevery little impact was observed in soil pH values (Table 1). The

Table 1Changes in soil pH, electrical conductivity and sodium content in response todifferent levels of salt stress.

Treatment pH# ECe Na (%)

0 mM 7.82 1.28d 0.035d

25 mM 7.95 2.61c 0.068c

50 mM 7.89 3.68b 0.098b

100 mM 8.02 5.36a 0.148a

# statistically non-significant at P0.05.

build-up of salinity level was so high in 100 mM NaCl treatment(ECe: 5.36 dS m�1; Na+ conc. 0.148%) that severe plant mortalitywas observed especially in susceptible genotypes (TMV 2 andNRCG 357) with distinctive visual difference in tolerant andsusceptible genotypes (Fig. 1). Due to higher plant mortality ofsensitive genotypes under 100 mM NaCl treatment, we hadrestricted further experiment up to 50 mM NaCl stress (i.e. 0, 25and 50 mM NaCl stress).

3.2. Salinity stress resulted in build-up severe oxidative stress anddecline in membrane stability

Salinity treatment (both 25 and 50 mM) resulted in significantreduction in membrane stability index (MSI) in all the genotypes(Fig. 2A). Genotypic difference was found to be more significant at50 mM NaCl treatment as compared 25 mM. The three tolerantgenotypes (Somnath, TPG 41, CS 240) showed �20% decline in MSI,while the sensitive genotypes (NRCG 357, TMV 2) showed �30%reduction under 50 mM NaCl treatment as compared to controlplants. To determine the levels of oxidative stress encountered bythe plants under different salinity level, we had estimated H2O2

and superoxide radical (SOR) content, and lipid peroxidation as ameasure of reactive oxygen species (ROS) load in these genotypes(Fig. 2B–D). The SOR content showed significant increase underboth 25 and 50 mM salt treatment, but the level of increase wasmore than double under 50 mM NaCl treatment as compared to25 mM (Fig. 2B). The genotype Somnath and TPG 41 showed leastincrease (�60%) in SOR content, while NRCG 357 recorded highestrise (200%) in response to 50 mM NaCl treatment.

Another major cellular ROS, H2O2 content was increased by �75and 200% at 25 and 50 mM NaCl treatment, respectively (Fig. 2C).Among the genotypes, TPG 41 showed least increase (55 and 110%,respectively at 25 and 50 mM treatment) in H2O2 content, whileNRCG 357 showed highest increase (>2-fold and >4.5-fold,respectively at 25 and 50 mM treatment). The lipid peroxidationmeasured in terms of TBARS content, increased with increasinglevel of salt stress. Among the genotypes, lipid peroxidation wassignificantly lower (10.99 mmol g�1 FW) in most tolerant genotype,Somnath, however it was found to be significantly higher(>14 mmol g�1 FW) in both the sensitive genotypes (Fig. 2D).

To confirm the ROS level in both control and salt stressed leaves,we had also carried out histochemical staining of leaves for in situlocalization of H2O2 and superoxide radical (O2

�) by DAB and NBTstaining, respectively (Fig. 3). DAB staining of representative leavesshowed very little dark spots in tolerant genotypes under stress,while the sensitive genotypes showed much darker staining evenat 25 mM NaCl treatment (Fig. 3A), thus confirming greater H2O2

load in the leaves of sensitive genotypes. Similarly, NBT stainingalso revealed relatively higher build-up of SOR in TMV 2 and NRCG357 as compared to tolerant genotypes under stress condition(Fig. 3B). These results are in complete agreement with spectro-metric detection of ROS load under saline condition andreconfirmed the fact that the level of oxidative stress was muchhigher in sensitive genotypes under similar level of salt stress. Tounderstand the differential ROS build-up observed in tolerant andsensitive genotypes, we further looked into the induction ofdifferent antioxidant machineries operating in peanut in responseto salt stress.

3.3. Changes in antioxidant enzyme activity at transcriptional andtranslational level

The present investigation showed salinity induced alteration inthe level of different antioxidant enzymes, though somewhatdifferentially in tolerant and sensitive genotypes. At enzyme level,the activity of SOD showed almost similar level of induction at both

Fig. 1. Differences in growth of peanut genotypes under control conditions and six weeks of different levels of salt stress.

Fig. 2. Changes in (A) membrane stability index, (B) superoxide radical and (C) hydrogen peroxide content, and (D) lipid peroxidation in peanut genotypes under differentlevels of salt stress.

K. Chakraborty et al. / Environmental and Experimental Botany 128 (2016) 79–90 83

25 and 50 mM NaCl treatment with slightly better response inSomnath as compared to NRCG 357 and TMV 2 (Fig. 4A,B). ExceptSomnath, there was no clear cut difference observed in inductionof SOD activity under salt stress in tolerant and sensitive

genotypes. When we compared the relative transcript abundanceof SOD at both the treatment levels, we found similarly lower levelof induction in some genotypes (mostly the tolerant ones), whilealmost no induction or slightly down regulation in others (mostly

Fig. 3. In situ determination of (A) hydrogen peroxide level by DAB staining and (B) superoxide radical load by NBT staining on peanut leaves under different levels of saltstress.

84 K. Chakraborty et al. / Environmental and Experimental Botany 128 (2016) 79–90

the sensitive ones) (Fig. 4C). The POD activity showed moreinduction under salt stress (Fig. 4D-F). Though the induction atenzyme level was relatively less, but a much profound increase inPOD transcript was observed under salt stress. The highest increasein POD activity was observed in both Somnath (1.8-fold) and Girnar1 (2.1-fold), which was in accordance with the changes attranscript level (3.9- and 3.2-fold increase, respectively) in thesegenotypes. The sensitive genotypes also showed some degree ofinduction in POD activity, but overall the magnitude of inductionwas relatively less in these genotypes.

In contrast to the SOD and POD enzymes, the activity of APXshowed induction neither at transcriptional nor at translationallevel (Fig. 5A–C). Although a slight increase (�25%) in APX enzymeactivity was observed in Somnath at 25 mM NaCl treatment, but itshowed no change at 50 mM salinity level as compared to controlplants. For rest of the genotypes, it either remained unchanged ordown-regulated with increasing levels of salt stress. Surprisingly,the APX transcript level was further down regulated in salt stressedplants irrespective of tolerant and sensitive genotypes. The GRactivity increased by �45% and �25% in Somnath and Girnar 1,respectively, in response to 50 mM NaCl stress, however it showedno induction or rather down-regulated in other genotypes undersalt stress (Fig. 5D,E). The relative abundance of GR transcript alsoshowed the exactly same pattern of induction as that of enzymeactivities for 50 mM NaCl treatment (Fig. 5F).

With imposition of salt stress, the CAT activity was found to beincreased in all genotypes studied (Fig. 6A,B). But unlike other fourantioxidant enzymes the induction pattern was almost reverse incase of CAT activity. Interestingly, here higher increase wasobserved in sensitive genotypes viz. TMV 2 (�50%) and NRCG357 (42%) than the tolerant ones viz. CS 240 (26%) and Somnath(33%). Unfortunately, we could not confirm this data withmolecular evidence as the custom made CAT specific primersdid not work after several rounds of attempts.

3.4. Changes in the level of non-enzymatic antioxidants

The levels of several key molecular antioxidants were changedin response to salt stress in the present study (Figs. 7 and 8). Amoderate but significant increase was observed in the level of freeproline under salt stress in all the genotypes (Fig. 7A). It showedhighest increase in Somnath (85%) and least in NRCG 357 under thehighest level of salt stress. Interestingly, another important cellularantioxidant, carotenoids showed completely reverse trend intolerant and sensitive genotypes. It increased by �30% in tolerantgenotypes (Somnath, TPG 41, CS 240); while there were 15–30%reduction in total carotenoid content observed in sensitive andmoderately tolerant genotypes (Fig. 7B).

Significant increase in total ascorbate pool was observed in thesalt treated plants in the present study (Fig. 8A–C). The increasewas mainly attributed to the sharp rise in the reduced form of

Fig. 4. Changes in superoxide dismutase activity (A) and its fold change (B), and relative transcript abundance (C) of SOD in peanut genotypes under different levels of saltstress. Changes in peroxidase activity (D) and its fold change (E), and relative transcript abundance (F) of POD in peanut genotypes under different levels of salt stress.

K. Chakraborty et al. / Environmental and Experimental Botany 128 (2016) 79–90 85

ascorbate (AsA) particularly in the tolerant genotypes in responseto salt stress (Fig. 8A). The highest increase in AsA content wasobserved in Somnath (�75%), while a mere �20% increase wasrecorded in both TMV 2 and NRCG 357. On the contrary, thedehydro-ascorbate (DHA) level was either increased nominally insome genotypes or remained unchanged in the most under saltstress (Fig. 8B). This increase in AsA content (may be because of denovo synthesis) in response to salt stress, ultimately resulted inmuch higher AsA/DHA ratio in salt treated plants, more so inSomnath, TPG 41 and Girnar 1 (Fig. 8C).

Significant changes in cellular glutathione pool (GSH and GSSG)in response to salt stress were recorded in the present study(Fig. 8D–F). The GSH content reduced by 22 and 41% at 25 and50 mM NaCl stress, respectively (Fig. 8D). Among the genotypes,the least reduction in GSH was observed in Somnath (29%), whileTMV 2 showed the highest reduction (52%). More severe reductionwas observed in GSSG content under salt stress in peanut. Unlike,GSH, the reduction in GSSG was �40% and 67% at 25 and 50 mMtreatment levels, respectively with distinctly higher reduction insensitive genotypes (Fig. 8E). Due to this, a shift in overall GSH/GSSG balance was observed under salt stress in the present study(Fig. 8F). The GSH/GSSG ratio was increased from 10.70 to 18.65 intolerant genotype Somnath, whereas the sensitive genotype NRCG357, showed much higher shift from 16.23 to 74.5 under 50 mM saltstress.

4. Discussion

Overall the results from the present study suggested moder-ately salt sensitive nature of peanut. Higher level of salt stress

(particularly 100 mM NaCl treatment) for a significant period oftime resulted in loss of complete plants stand in sensitivegenotypes (Fig. 1), which made impossible for us to comparegenotypes at this stress level, hence up to 50 mM NaCl treatmentadjudged to be the sufficient level of salt stress in order to getcontrasting responses from sensitive and tolerant peanut geno-types. The cultivated peanut, being allo-tetraploid (AABB genome)in nature, has relatively broader genotypic background consistingof two distinct botanical group viz. spreading or semi-spreadingtype Virginia genotypes (Arachis hypogaea sub sp. hypogaea) anderect or bunch type Spanish genotypes (Arachis hypogaea sub sp.fastigiata) (Moretzsohn et al., 2004). In the present study, wedeliberately chose to include sensitive and tolerant genotypes fromboth the botanical groups to nullify genotypic background effect onthe stress tolerance mechanism of peanut (see supplementarytable S1 for genotypic details). Indeed we did not observed anysuch effect on stress tolerance mechanism of peanut, rather themechanism varied between tolerant and sensitive genotypes andperhaps with the level of stress, which are discussed in details inthe following sections.

4.1. Differential ROS load may associate with salt sensitivity in peanut

Salt tolerance is a complex trait that involves number ofmechanisms to counteract osmotic and ionic effects of salt stress(reviewed in Munns and Tester, 2008). Detailed knowledge on eachof the salt tolerance mechanism is of utmost importance fordeveloping salt tolerant crop. Water deficit condition created dueto osmotic effect of salt stress often leads to production of reactiveoxygen species (ROS) viz. superoxide (O2�), hydrogen peroxide

Fig. 5. Changes in ascorbate peroxidase activity (A) and its fold change (B), and relative transcript abundance (C) in APX in peanut genotypes under different levels of saltstress. Changes in glutathione reductase activity (D) and its fold change (E), and relative transcript abundance (F) of GR in peanut genotypes under different levels of salt stress.

Fig. 6. Changes in catalase activity (A) and its fold change (B) in peanut genotypesunder different levels of salt stress.

86 K. Chakraborty et al. / Environmental and Experimental Botany 128 (2016) 79–90

(H2O2), hydroxyl radical (�OH) and singlet oxygen (1O2) in order tochannelize excess reducing power generated due to slowing downof dark reaction of photosynthesis (Hsu and Kao, 2003; Aboga-dallah, 2010). Several studies showed generation of significantamount of ROS and oxidative stress mediated toxic effects of NaClon legumes (Hernández et al., 2000) and other vascular plants (Leeet al., 2001; Mittova et al., 2004). In the present study, we foundsignificant reduction of membrane stability and production of bothSOR and H2O2, and lipid peroxidation in peanut genotypes undersalt stress. The level of ROS production and lipid peroxidation wasmuch higher in sensitive genotypes (NRCG 357 and TMV 2) ascompared to the tolerant ones (Fig. 2). The same pattern of ROSbuild-up was confirmed by in situ histochemical staining of peanutleaves, which suggested a greater ROS load in sensitive genotypesunder salt stress (Fig. 3). Thus from the present fact it is clear thatunder the similar level of salt stress the sensitive peanut genotypeswere accumulating more ROS in metabolically active leaf tissues,while the tolerant ones (Somnath, TPG 41, CS 240) were capableenough to keep the ROS level to a relative lower level.

4.2. Simultaneous induction of all the antioxidant enzymes does notnecessarily require for salt tolerance in peanut

To find out the reason for such differential ROS accumulation,we investigated the changes in the level of major antioxidantenzymes in these genotypes under salt stress, which were thoughtto be up-regulated under stress to bring down the harmful ROSload in cascade of reactions (Apel and Hirt, 2004; Bor et al., 2003).However, several other reports suggested that efficient antioxidant

Fig. 7. Changes in proline (A) and total carotenoids (B) content in peanut genotypesunder different levels of salt stress.

Fig. 8. Changes in reduced ascorbate (A), dehydro-ascorbate (B), reduced to oxidized

reduced to oxidized glutathione ratio (F) in peanut genotypes under different levels of

K. Chakraborty et al. / Environmental and Experimental Botany 128 (2016) 79–90 87

enzyme activity does not necessarily mean strong up-regulation ofthe full set of antioxidant enzymes (reviewed in Abogadallah,2010). Cavalcanti et al. (2004), reported antioxidant defensemediated by SOD, CAT and POD activities was not required for salttolerance in cowpea, while increased activities of POD, CAT and GRwas reported to increase salt tolerance in French bean (NageshBabu and Devaraj, 2008). In the C4 barnyard grass, little role of SODactivity was reported as scavenger of oxidative stress under salinecondition, whereas peroxidases was found to play major role infine regulation of ROS balance to impart salt tolerance (Aboga-dallah et al., 2010). Similarly, the increased activities of Halliwell-Asada pathway enzymes (APX, GR, MDHAR and DHAR) werereported in wheat, while Cu/Zn-SOD activity remained constantunder salt stress (Hernández et al., 2000). Like glycophytes, thescenario remains similar for halophytes, where Parida et al. (2004)reported up regulation of SOD, POD, APX and GR but downregulation of CAT in response to salt stress in mangroves.

Thus taking clues from the existing evidences found fromdiverse group of plants, it is clear that the ROS detoxificationmechanism may not be limited to simple up-regulation of full setof antioxidant enzymes, rather selective induction of one or othercomponent has been intriguingly associated with salt tolerance,which may differ from species to species as well as with intensityof stress. Though, in the present study we found induction of SODactivity under salt stress, but at the transcript level, relative mRNAabundance did not show much induction of SOD transcript withincreasing salt level. More importantly, the induction pattern ofSOD (both transcriptional and translational) did not differ

ascorbate ratio (C), reduced glutathione (D), oxidized glutathione content (E) and salt stress.

88 K. Chakraborty et al. / Environmental and Experimental Botany 128 (2016) 79–90

significantly between tolerant and sensitive genotypes (Fig. 4A–C),suggesting nominal role of SOD in overall salt tolerance in peanut.It further suggested that probably the little change in SOD level issufficient enough to carry out initial scavenging reaction (conver-sion from O2� to H2O) and thus variation at this step is perhapsmore substrate dependent rather related to salt-sensitivity ofpeanut genotypes. Our findings are in agreement with someprevious reports, which suggested higher SOD activity was notrelated to salt tolerance in barnyard grass, rather it might be due tosecondary effect of higher SOR (O2�) content, which provoked SODinduction under salt stress (Abogadallah et al., 2010). On thecontrary, Lechno et al. (1997) reported NaCl treatment did notaffect the activity of SOD in cucumber plants, while Pan et al.(2006) observed initial drop in SOD activity followed by itsrecovery to control level in response to salt and drought stress inGlycyrrhiza; whereas some researcher correlated increased SODactivity with plant’s ability to tolerate salt stress in mulberry, chickpea, tomato etc. (Ben Amor et al., 2006; Sekmen et al., 2007).

As a whole, plant has a coordinated mechanism of ROSdetoxification, where SOD thought to act as the first line ofdefense triggering dismutation reaction to convert SOR (O2�) toH2O2, generated from photosynthetic and respiratory electronleakage mostly under unfavorable conditions. Now at cellular level,thus produced H2O2 could be channelized to H2O production by atleast three major enzymatic paths by using various reducingsubstances. These include POD, CAT and APX-GR driven Halliwell-Asada pathway (reviewed in Gill and Tuteja, 2010). As both PODand APX have much stronger affinity for H2O2 than CAT, so it isbelieved that both POD and APX are associated with H2O2

scavenging under lower concentration, while the en masse H2O2

scavenging is facilitated by CAT (Asada, 1999; Mittler, 2002). Amuch stronger induction of POD (both transcriptional andtranslational level) under salt stress, was observed in the presentstudy, which was more significant in tolerant genotypes (Fig. 4D–F), while the activities and relative transcript abundance of APX didnot up-regulated under salt stress in either tolerant or sensitivegenotypes (Fig. 5). In contrast to POD activity, CAT showedcompletely opposite trend of induction by showing relativelyhigher increase in sensitive genotypes under salt stress ascompared to the tolerant ones (Fig. 6). Looking at the evidences

Fig. 9. Proposed hypothetical network showing selective induction of ROS detoxificatiodifferent enzymes of antioxidant defense network are represented by different geometricinduction in response to salt stress.

unfolded from the present study, it is clear that at relatively lowerlevel of H2O2 concentration, POD acted as the major detoxifyingenzyme in peanut under salt stress, which may well associatedwith fine regulation of H2O2 in both tolerant and sensitivegenotypes. Additionally, CAT also played important role insupplementing POD activity in sensitive genotypes, where therewas relatively higher H2O2 load. Cavalcanti et al. (2004) reportedinduction of POD activity in response to salt stress, althoughchanges in both CAT and SOD were non-significant in cowpea.Abogadallah et al. (2010) reported induction of POD and APX-GR asthe major H2O2 scavenger at relatively low H2O2 concentration andacted as fine regulator for ROS balance in barnyard grass, while CATalthough could able to detoxify much higher level of H2O2, butcould not maintain balanced levels of ROS under salt stress. But inthe present study, we did not find much role of APX-GR pathway inROS detoxification in peanut under salt stress. This could be due tovery specific adaptive response of peanut under salt stress, whereit prefers to induce single enzyme (POD in this case) for H2O2

scavenging at low H2O2 concentration and thus saving significantmetabolic investment required to up-regulate at least fourenzymes of APX-GR driven Halliwell-Asada pathway for H2O2

detoxification.

4.3. Non-enzymatic antioxidants supplement efficient enzymatic ROSdetoxification system in peanut under salt stress

Apart from the enzyme driven antioxidant defense system, lowmolecular weight antioxidant viz. ascorbate, glutathione, carote-noids etc. play crucial role in counter balancing oxidative stress andregulation of the cellular ROS homeostasis in plants (Schafer et al.,2002; Ashraf, 2009)). Carotenoids, a lipophilic organic compound,besides absorbing light from visible spectra (450–570 nm) andpassing on to the reaction center, provides necessary photo-protection to the chlorophyll molecules surrounding the photo-system (Taiz and Zeiger, 2006). Different types of plant carotenoidsprevent the formation of ROS by quenching the triplet state ofchlorophyll molecules near photosynthetic reaction center (Fyfeet al., 1995). Compatible solutes such as proline accumulated inresponse to salt or water deficit stress primarily for osmoticadjustment, but also act as low molecular weight cellular

n pathway in tolerant and sensitive genotypes of peanut under salt stress, where shapes with their respective area giving an apparent indication about magnitude of

K. Chakraborty et al. / Environmental and Experimental Botany 128 (2016) 79–90 89

antioxidant (Ashraf and Foolad, 2007). When applied exogenouslyproline could reduce the level of ROS in Arabidopsis roots,indicating ROS scavenging potential of proline (Cuin and Shabala,2007). Besides its role as ROS scavenger, an increased endogenousproline level might also helped as osmoprotectant to safeguardcellular macromolecular structure and improved salt-tolerance inthese genotypes.

Our results showed, rise in total ascorbate (mainly the reducedform) and proline content in salt stressed peanut, while thecarotenoid level was only increased in tolerant genotypes anddeclined in the sensitive ones (Figs. 7 and 8). The increased activityof APX-GR pathway uses ascorbate as reducing agent and thusconverts it to oxidized form, resulting in overall decrease in totalsoluble ascorbate pool mainly due to the loss of the reduced formof ascorbate (Hernández et al., 2000). But we observed theopposite trend in the ascorbate level, which may be attributed tothe maintenance of reduced form of ascorbate and/or de novosynthesis of it as a salinity induced response in peanut, whichultimately led to higher AsA content and increased AsA/DHA ratioin salt treated plants (particularly in tolerant genotypes), followedby its minimal utilization in APX-GR pathway. This resultsuggested a possibility of direct ROS scavenging role of ascorbatein peanut rather than its utilization in APX driven ROS detoxifica-tion route. Similar increase in reduced form ascorbate and higherAsA/DHA ratio was previously reported in salinity and other abioticstresses (Athar et al., 2008; Locato et al., 2008). On the other hand,the glutathione pool (both GSH and GSSG) was reduced under saltstress with relatively lower reduction observed in the tolerantgenotypes (Fig. 8D–F). As reported previously, the decline inglutathione contents may be wholly or partly related to reducedrate of GSH synthesis and/or increased rates of degradation or GSHtransport to other plant organs (Herschbach et al., 1998;Hernández et al., 2000).

5. Conclusion

From the present study we can conclude that salt tolerant andsensitive peanut genotypes behaved differently under salt stress interms of ROS accumulation and oxidative stress toleranceattributes, with no obvious difference observed in relation totheir genotypic character. Lower ROS load in tolerant genotypesmay be attributed to better antioxidant defense (both enzymaticand non-enzymatic) capacity and cellular antioxidant pool.Interestingly, from the present study, it seems that peanutgenotypes does not necessarily require simultaneous inductionof full set of antioxidant enzymes for salt tolerance, rather POD wasfound to be the major H2O2 detoxifying enzyme in tolerant peanutgenotypes, where there is relatively lesser H2O2 load and it wassupplemented by CAT activity at relatively higher H2O2 load (insensitive genotypes). Minimal induction of SOD (with no signifi-cant difference between tolerant and sensitive genotypes) andalmost no induction of APX and GR activities suggested nominalassociation of these enzymes with overall salt tolerance in peanut(Fig. 9).

Conflict of interest

The authors declare that they have no conflict of interest.

Author contributions

KC conceptualized the whole study, KC and ALS designed theexperiment, PVZ and NG performed pot experiments. KC and NGanalysed the data and done the statistical analysis, KC, SKB and ALSdrafted the manuscript. KC and SKB carried out the molecular

work, NG and PVZ performed the physiological and biochemicalstudies.

Acknowledgements

Authors gratefully acknowledge the support from Director,ICAR-DGR for the present study. We also acknowledge the supportof Dr. D Bhaduri for carrying out soil analysis, Dr. SK Bera forproviding the seeds of ‘CS 240’ and Dr. K Patel for his help renderedin molecular works.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.envexpbot.2016.05.001.

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