ORIGINAL PAPER

Arbuscular mycorrhizal symbiosis modulates antioxidantresponse in salt-stressed Trigonella foenum-graecum plants

Heikham Evelin & Rupam Kapoor

Received: 13 June 2013 /Accepted: 16 September 2013 /Published online: 11 October 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract An experiment was conducted to evaluate the in-fluence ofGlomus intraradices colonization on the activity ofantioxidant enzymes [superoxide dismutase (SOD), catalase(CAT), peroxidase (PX), ascorbate peroxidase (APX), andglutathione reductase (GR)] and the accumulation ofnonenzymatic antioxidants (ascorbic acid, α-tocopherol, glu-tathione, and carotenoids) in roots and leaves of fenugreekplants subjected to varying degrees of salinity (0, 50, 100, and200 mM NaCl) at two time intervals (1 and 14 days aftersaline treatment, DAT). The antioxidative capacity was corre-lated with oxidative damage in the same tissue. Under saltstress, lipid peroxidation and H2O2 concentration increasedwith increasing severity and duration of salt stress (DoS).However, the extent of oxidative damage in mycorrhizalplants was less compared to nonmycorrhizal plants. The studyreveals that mycorrhiza-mediated attenuation of oxidativestress in fenugreek plants is due to enhanced activity ofantioxidant enzymes and higher concentrations of antioxidantmolecules. However, the significant effect of G. intraradicescolonization on individual antioxidant molecules and en-zymes varied with plant tissue, salinity level, and DoS. Thesignificant effect of G. intraradices colonization onantioxidative enzymes was more evident at 1DAT in bothleaves and roots, while the concentrations of antioxidant mol-ecules were significantly influenced at 14DAT. It is proposedthat AM symbiosis can improve antioxidative defense sys-tems of plants through higher SOD activity in M plants,facilitating rapid dismutation of O2

- to H2O2, and subsequentprevention of H2O2 build-up by higher activities of CAT,APX, and PX. The potential of G. intraradices to ameliorate

oxidative stress generated in fenugreek plants by salinity wasmore evident at higher intensities of salt stress.

Keywords Antioxidative enzymes . Arbuscular mycorrhiza .

Lipid peroxidation . Nonenzymatic antioxidants . Salt stress

Abbreviations

APX Ascorbate peroxidaseCAT CatalaseDAT Days after treatmentDoS Duration of stressGR Glutathione reductaseM MycorrhizalNM NonmycorrhizalPX PeroxidaseSOD Superoxide dismutase

Introduction

Land salinization is a major global problem as it negativelyaffects plant growth and productivity causing a threat to foodsecurity (Reitz and Haynes 2003; Azevedo Neto et al. 2006;Ashraf 2009; Evelin et al. 2009; Abogadallah 2010; Bothe2012; Porcel et al. 2012; Ruiz-Lozano et al. 2012).Inappropriate agricultural practices, poor soil management,limited rainfall, high temperature, and high evapotranspirationrates (Canterll and Linderman 2001; Azevedo Neto et al.2006; Chen et al. 2011) have largely contributed to increasingsalinity with each passing year (Chen et al. 2011). It isprojected that by the middle of the 21st century, 50 % ofarable land will become saline (Wang et al. 2003). High levelsof salt in soil or irrigation water affect the metabolismof the plant, causing hyperionic and hyperosmotic stress

H. Evelin : R. Kapoor (*)Applied Mycology Laboratory, Department of Botany,University of Delhi, Delhi 110 007, Indiae-mail: kapoor_rupam@yahoo.com

Mycorrhiza (2014) 24:197–208DOI 10.1007/s00572-013-0529-4

(Vaidyanathan et al. 2003; Ramoliya et al. 2006; Porcelet al. 2012). These injurious osmotic effects and ionic toxicitylead to the generation of secondary stress, known as “oxidativestress,” a condition in which the equilibrium between the pro-duction of reactive oxygen species (ROS) and the quenchingactivity of antioxidants is disturbed (Gill and Tuteja 2010).Oxidative stress is a consequence of uncoupled pathways inthe metabolism of plants transferring high-energy state electronsto molecular oxygen to form ROS (Gill and Tuteja 2010). ROSinclude singlet oxygen (1O2), superoxide anion (O2

-), hydrogenperoxide (H2O2), and hydroxyl radical (OH). ROS perturbvarious cellular functions by damaging nucleic acids, oxidizingproteins, and causing lipid peroxidation (Foyer and Noctor2005). Plants have two antioxidative systems to counter theeffects of ROS: enzymatic and nonenzymatic scavenging sys-tems. Enzymatic scavenging systems include superoxidedismutase (SOD), catalase (CAT), peroxidase (PX), ascorbateperoxidase (APX), and glutathione reductase (GR). Antioxidantmolecules, such as ascorbic acid, glutathione, α-tocopherol, andcarotenoids, also play important roles in the removal of toxic by-products of O2 (Gill and Tuteja 2010). In fact, plant tolerance tosalt stress has been associated with the induction of antioxidantenzymes and reduction of oxidative damage (Sekmen et al.2007; Garg and Manchanda 2009; Turkan and Demiral 2009;Manchanda and Garg 2011; Ruiz-Lozano et al. 2012).

Arbuscular mycorrhizal fungi (AMF) form an integral com-ponent of the soil biota and their colonization of host plantsimproves plant tolerance to salinity stress by preventing ionictoxicity and hyperosmotic stress (Evelin et al. 2009; Porcel et al.2012). In doing so, AMF improve nutrient acquisition, ionicbalance, water uptake, and osmoregulation and prevent ultra-structural damage in plants under saline stress (Garg andManchanda 2009; Hajiboland et al. 2010; Abdel Latef andChaoxing 2011; Evelin et al. 2012, 2013). A few studies havedemonstrated that AMF inoculation can prevent/limit salt-induced oxidative stress in host plants (ZhongQun et al. 2007;Garg and Manchanda 2009; Hajiboland et al. 2010; Wu et al.2010; Manchanda and Garg 2011; Estrada et al. 2013). Undersalt stress, plants colonized byAMF have shown higher activityof antioxidant enzymes such as SOD, CAT, PX, APX, and GR(ZhongQun et al. 2007; Garg andManchanda 2009; Hajibolandet al. 2010;Wu et al. 2010; Manchanda and Garg 2011; Estradaet al. 2013). The latter authors proposed that higher antioxidantenzyme activity in mycorrhizal (M) plants helps in rapid andefficient removal of excess ROS. However, reports on theinfluence of AM symbioses on antioxidant system under stressconditions are not consistent. Studies have reported increase(ZhongQun et al. 2007; Garg andManchanda 2009; Hajibolandet al. 2010), no change (Ghorbanli et al. 2004; Borde et al.2011), or decrease (Abdel Latef and Chaoxing 2011) in anti-oxidant enzyme activity in M plants under stress conditions.

Antioxidant enzyme activities vary with the plant organ(Rios-Gonzalez et al. 2002) as well as the severity and duration

of stress (DOS; Abogadallah 2010). Despite roots being theorgan which harbors M fungi and that they are in direct contactwith saline soil, antioxidant enzymes have rarely been studiedin roots. Also, most of the studies involve a single samplingtime (Abogadallah 2010). Furthermore, the influence of AMsymbioses on the accumulation of nonenzymatic antioxidants,such as ascorbic acid, α-tocopherol, glutathione, and caroten-oids in host plants, have been poorly studied (Ruiz-Lozanoet al. 2012). Although ROS accumulation largely depends onROS production and scavenging (Miller et al. 2010), antioxi-dant enzyme activities are rarely correlated with ROSmeasure-ments (Ruiz-Lozano et al. 2012). Therefore, in order to obtain aclearer picture of the influence of AM symbioses during saltstress on antioxidant accumulation in plant, a study wasperformed (i) to evaluate the activity of ROS scavenging en-zymes (SOD, CAT, PX, APX, and GR) and accumulation ofnonenzymatic antioxidants (ascorbic acid, glutathione, carot-enoids, and α-tocopherol) in both roots and leaves subjected tovarying degrees of salinity at two time intervals; and (ii) tocorrelate changes in antioxidative capacity with levels of H2O2

and oxidative damage in the same tissue.

Materials and methods

Experimental design, growth conditions, and soil

A pot experiment was conducted in the Botanical Garden ofDepartment of Botany, University of Delhi, Delhi, India.During the experimental period, the temperature ranged be-tween 24 and 29 °C and relative humidity between 31 % and67 %. The soil used was sandy loam and contained P 3,100 mg kg-1, K+ 520 mg kg-1, Ca2+ 2,300 mg kg-1, Mg2+ 4,600 mg kg-1, Na+ 320 mg kg-1, Cu 4.43 mg kg-1, Mn2+

200 mg kg-1, and Zn2+ 55.52 mg kg-1 (Allen 1989). The soilwas sieved and mixed with an equal proportion of sand. Thesand–soil mixture was autoclaved for 15 min at 121 °C and15 psi to destroy the existing AM propagules.

The experiment was a randomized complete block designwith two factors: AMF inoculation and salinity levels. Therewere four levels of salt treatment (0, 50, 100, and 200 mMNaCl) and two M conditions (AMF-inoculated and AMF-noninoculated). Hence, there were eight treatments (2×4)and each treatment was replicated three times.

Plant material

Fenugreek (Trigonella foenum-graecum L.) var. Pusa EarlyBunching was used in the study. Seeds were obtained fromNational Seeds Corporation, New Delhi, India. Six surface-disinfected seeds of fenugreek were sown in each plastic potcontaining 2 kg of an autoclaved sand:soil mix (1:1). Afteremergence, seedlings were thinned to three per pot. Since

198 Mycorrhiza (2014) 24:197–208

fenugreek is a dry land crop with lowwater requirement (Basuet al. 2004), plants were watered with autoclaved tap watertwice a week.

AMF inoculum and inoculation

Glomus intraradices (Schenck and Smith) inoculum (acces-sion number CMCCWep319) was procured from The Energyand Resources Institute (TERI, New Delhi, India) and propa-gated as soil-based open cultures in sterile a sand:soil mixture(1:1) using Sorghum bicolor L. and Trigonella foenum-graecum L. var. Pusa Early Bunching as trap plants, grownalternatively (Kapoor et al. 2002). The cultures weremaintained under natural conditions of temperature, lightand humidity for one year. The trap plants were cut at thebase, the roots chopped into small pieces and mixed with thesoil mass of the culture pots. AMF inoculum (10 g) containedabout 60–70 infectious propagules. Propagule infectivity wastested according to the method of Sharma et al. (1996).

At the time of sowing of fenugreek seeds, half of the potswere inoculated with 50 g of AMF inoculum at a depth of 3 cmand mixed with soil. The uninoculated control plants receivedthe same amount of autoclaved M inoculum together with afiltrate of an inoculum suspension in water, to reintroduce thenative microbial community except for M propagules. The inoc-ulum suspensionwas prepared by suspending 50 g inoculum soilin 50 ml distilled water and filtered using Whatman No. 1.

Saline stress

After 30 days of sowing, plants were subjected to four levels ofNaCl stress (0, 50, 100, and 200 mM NaCl). Plants wereirrigatedwith one of eachNaCl solution (50ml pot-1) for 7 daysand a total volume of 350 ml of saline solution was added toeach pot in the experiment. Upon addition of saline solutions,the electrical conductivity (EC) of saturated soil extracts in-creased to 0.019, 0.21, 0.52, and 0.91 S m-1 in the 0, 50, 100,and 200mMNaCl treatments, respectively. Addition of 350 mlof 50, 100, and 200 mM NaCl resulted in the addition of saltamounting to 8.75, 17.5, and 35 mM kg-1 soil, respectively,after 7 days of saline treatment. The plants were irrigated withautoclaved tap water twice a week, and the EC of the soilextract was monitored using a conductivity meter (DecibelDB-1401) and adjusted once in 15 days with the respectiveNaCl solution. Plants were sampled on the 1st and 14th dayafter saline treatment (DAT) for analyses. AMF colonizationand plant biomass were measured at final harvest.

Measurement of oxidative damage

Lipid peroxidation in leaves and roots of M and NM fenu-greek plants was detected according to Heath and Packer(1968) by measuring the concentration of malondialdehyde

(MDA). The amount of MDAwas calculated using extinctioncoefficient of 155 mM-1 cm-1 and expressed as nmolMDA g-1

fresh weight.H2O2 concentrations in leaves and roots were determined

according to Velikova et al. (2000). Plant tissue (0.07 g) washomogenized with 5 ml 0.1 % (w/v) trichloroacetic acid in anice bath. The homogenate was centrifuged at 12,000×g for15 min and the supernatant was used for determination ofH2O2 concentration. The assay mixture contained 0.5 ml su-pernatant, 0.5 ml 10 mM phosphate buffer (pH 7.0), and 1 ml1 M KI. The assay mixture was kept in dark for 1 h and theabsorbance was read at 390 nm. The concentration of H2O2

was determined from a standard curve and expressed as μgH2O2 g

-1 fresh weight.

Measurement of antioxidant molecules

The concentrations of ascorbic acid and glutathione in leavesand roots were determined according to Wu et al. (2006).Samples (0.5 g) were powdered in liquid N2 and homogenizedin 5 % trichloroacetic acid. The homogenate was centrifugedat 12,000 rpm for 15 min at 4 °C. The supernatant was usedfor determination of ascorbic acid and glutathione concentra-tions. Ascorbic acid concentration was determined in an assaymixture containing 0.5 ml supernatant, 0.1 M phosphate buff-er pH 7.7, 10 % (w/v) trichloroacetic acid, 44 % (w/v) H3PO3,4 % (w/v) 2, 2′-bipyridyl and 3 % (w/v) FeCl3. The mixturewas incubated at 37 °C for 1 h, cooled to room temperature,and absorbance read at 525 nm. The concentration of ascorbicacid was calculated from a standard curve of ascorbic acid andexpressed as μg ascorbic acid g-1 fresh weight. Glutathioneconcentration was determined in a reactionmixture containing1 ml supernatant, 0.1 M phosphate buffer (pH 7.7), and0.60 mM 5,5′-dithiobis (2-nitro-benzoic acid). The absor-bance of the mixture was read at 412 nm. The concentrationof glutathione in the samples was calculated using a standardcurve prepared with glutathione and expressed as μg glutathi-one g-1 fresh weight.

The concentration (μg g-1 fresh weight) of α-tocopherolwas estimated according to Sadasivam and Manickam (2008)with slight modifications as described elsewhere (Evelin et al.2013). Carotenoids in leaves were extracted in dimethyl sulf-oxide and its concentration (mg g-1 fresh weight) was calcu-lated using the formula given by Arnon (1949).

Measurement of antioxidant enzymes activities

Antioxidant enzymes were extracted as follows: a fresh leaf orroot sample (1 g) was ground to a fine powder in liquidnitrogen and homogenized with a mortar and pestle in 4 mlof ice-cold 0.2 M phosphate buffer (pH 7.8) containing0.1 mM EDTA. The homogenate was centrifuged at 12,000 rpm for 20 min, and the supernatant was used as a crude

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enzyme source. An aliquot of the extract was used to deter-mine protein concentration by the method of Bradford (1976)using bovine serum albumin as the standard. All the opera-tions were performed at 4 °C, and the spectrophotometricanalyses were conducted on a UV/Vis spectrophotometerBeckman Coulter DU®730.

SOD activity was determined following the method de-scribed by Elavarthi and Martin (2010). The reaction mixtureconsisted of 50 mM phosphate buffer (pH 7.8) containing2 mM EDTA, 9.9 mM L-methionine, 55 μM NBT, 0.025 %Triton-X 100, and 50 μl of enzyme. Riboflavin (1 mM) wasadded last as a source of O2

-, and the reaction was initiated byilluminating the samples under 15 W fluorescent tube.Absorbance of the sample was measured immediately at560 nm after the reaction was stopped. One unit of enzymeactivity was defined as the amount of enzyme required tobring about 50 % inhibition of the rate of NBT reductionmeasured at 560 nm and expressed as nkatals mg-1 proteinfresh weight. CAT (EC 1.11.1.6) activity was determinedaccording to Aebi and Lester (1984) by monitoring the de-crease in absorbance at 240 nm due to decomposition of H2O2

in a 3 ml assay mixture containing 2 ml leaf extract and10 mM H2O2. The enzyme activity was expressed as μkatalsmg-1 protein fresh weight. APX (EC 1.11.1.11) activity wasdetermined by monitoring the oxidation of ascorbate as de-scribed byNakano and Asada (1987) and expressed asμkatalsmg-1 protein fresh weight. PX (EC (EC 1.11.1.7) activity wasdetermined according to Narwal et al. (2009) by monitoringthe oxidation of guaiacol at 412 nm for 5 min. The enzymeactivity was expressed as μkatals mg-1 protein fresh weight.GR (EC 1.6.4.2) activity was measured according to Smithet al. (1989) by monitoring the reduction of DTNB to TNB byglutathione in the reaction at 412 nm. The enzyme activitywas expressed as μkatals mg-1 protein fresh weight.

Statistical analysis

Data were subjected to correlation analysis (p <0.05) and three-way analysis of covariance (ANCOVA) with M inoculation,NaCl treatment, DoS, and interactions among them as sourcesof variation. In order to determine significance of means,Duncan’s multiple-range test (DMRT) was performed usingSPSS 14.0 statistical program (SPSS, Chicago, IL, USA).

Results

AMF colonization and plant biomass

Salt stress affected the ability of G. intraradices to colonizeroots of T. foenum-graecum . There was a decrease in thepercent AMF colonization with increase in salinity (68–32 %;Table 1). Increased salinity reduced lengths and dry weights of

both shoot and root of M as well as nonmycorrhizal (NM)plants. However, M plants showed better growth and biomassthan their corresponding NM plants at each level of salinity(Table 1).

Oxidative damage

Under salt stress, lipid peroxidation (measured as MDA) inleaves and roots of M and NM plants showed a linear increasewith corresponding increases in the level of salt in the soil(Fig. 1A–D). However, lipid peroxidation levels in both tis-sues of M as well as NM plants were higher at 14DATcompared to 1DAT. This was confirmed by the significantindependent effect of NaCl and DoS as well as their interac-tion (Table 2). The level of lipid peroxidation in both leavesand roots were approximately same. By and large, M plantsshowed lower levels of lipid peroxidation than the corre-sponding NM plants at each salt level. Initially (1DAT),significant differences in MDA concentrations between Mand NM plants were evident only at higher NaCl levels (at200 mM NaCl in leaves and 100 and 200 mM NaCl in roots);however, at a later stage of stress (14DAT), M plants showedsignificantly lower MDA levels in both tissues at all levels ofsalinity (Fig. 1A–D).

With increase in salt stress, there was a gradual increase inH2O2 concentrations in M and NM fenugreek plants at both1DATand 14DAT (Fig. 2A–D). The H2O2 level was higher inplant tissues at 14DAT as compared to plants harvested at

Table 1 Influence of NaCl andG. intraradices inoculation on shoot androot dry weights and percent colonization in nonmycorrhizal (NM) andmycorrhizal (M) fenugreek plants

TreatmentNaCl (mM)

AMstatus

Shoot dryweight (g)

Root dryweight (g)

Percentcolonization

0 NM 0.4c 0.08c

M 0.91e 0.10d 68d

50 NM 0.3b 0.06b

M 0.43d 0.09cd 52c

100 NM 0.3b 0.05b

M 0.32bc 0.06b 44b

200 NM 0.2a 0.03a

M 0.3b 0.05b 32a

Significance

NaCl ** ** ***

AMF ** ** ***

NaCl×AMF NS NS ***

Values are means of three replicates (±SD). Different letters indicatesignificant differences at p <0.05

NS not significant***P<0.001, **P <0.01, *P <0.05

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1DAT. Moreover, the values were higher in leaves than rootsat 14DAT. In roots, the increase in H2O2 concentration wasalso influenced by the interactions NaCl×AMF, NaCl×DoS,and NaCl×AMF×DoS (Table 2). Regardless of the intensity ofsalinity, M plants displayed lower H2O2 concentration thancorresponding NMplants at both 1DATand 14DAT; however,the differences were not significant for roots.

Antioxidant molecules

M colonization positively influenced the concentration of an-tioxidant molecules—ascorbic acid,α-tocopherol, glutathione,and carotenoids in fenugreek plants. The significant increase inascorbic acid level in M plants was evident at higher intensitiesof stress (100 and 200 mMNaCl), except for leaves at 14DAT(Fig. 3A–D). Three-way ANCOVA demonstrated significantindependent effects of NaCl and AMF as well as their interac-tions on ascorbic acid concentrations (Table 2). The concen-trations of α-tocopherol, glutathione, and carotenoids werehigher in M plants than NM plants at all levels of salinityand DoS (Figs. 4A–D, 5A–D, and 6A–B, respectively). Thethree independent factors, NaCl, AMF, and DoS, and theinteractions between and among them displayed significanteffects on these antioxidant molecules (Table 2). While effectof G. intraradices colonization on α-tocopherol concentrationwas evident at higher salinity levels (100 and 200 mM NaCl),the concentration of glutathione and carotenoids were signifi-cantly higher in M plants even when not subjected to saltstress. Overall, the effect of G. intraradices colonization on

the concentration of antioxidant molecules was more evidentwith increase in DoS.

Three-way ANCOVA revealed that the correlation betweenthe concentration of α-tocopherol and the level of lipid perox-idation was significantly influenced by NaCl, AMF, DoS, andtheir interactions (Table 2). There was also a strong positivecorrelation between α-tocopherol and ascorbic acid in leavesand roots at both sampling times (r =0.97 at 1DAT leaf; r =0.86 at 14DAT leaf; r =0.98 at 1DAT root; and r =0.95 at14DAT root). Furthermore, the correlation between these twowas significantly influenced by NaCl, AMF, DoS, and theirinteractions (Table 2).

Antioxidant enzymes activities

G. intraradices colonization had a positive effect on SODactivity in fenugreek plants under saline as well as nonsalineconditions (Tables 3 and 4). Exposure to salt stress resulted inan increase in SOD activity in leaves and roots in both M andNM plants, which was much greater at 14DAT compared tovalues at 1DAT. This was confirmed by the significant effectsof NaCl and DoS independently as well as the interactionbetween them (Table 2). However, SOD activity in roots ofboth M and NM plants were higher than in leaves at all times.Interestingly, three-way ANCOVA also revealed significanteffects of NaCl, AMF, DoS, and their interactions on thecorrelation between H2O2 concentrations and SOD activity.

CATactivity in leaves of NM plants increased with increas-ing NaCl level and time of exposure to salt stress (Table 3). In

Fig. 1 Influence of NaCl andGlomus intraradices inoculationon leaf (A–B) and root (C–D)malondialdehyde (MDA)concentration (nmol g-1 freshweight) in Trigonella foenum-graecum at 1DAT and 14DAT.Values represent mean ofreplicates; ±SD. Different lettersindicate significant differences atp <0.05. DAT days aftertreatment, M mycorrhizal, NMnonmycorrhizal

Mycorrhiza (2014) 24:197–208 201

contrast, although root CAT activity increased over time, itdeclined with increase in the NaCl level (Table 4). At theinitial stage of stress, CAT activity in M plants was signifi-cantly higher compared to NM plants. However, these differ-ences were not significant with increase in DoS. Three-wayANCOVA revealed a significant influence of NaCl, AMF andDoS, and their interactions (Table 2) on the correlation be-tween CAT activity and H2O2 concentration. However, therewas no significant effect of AMF×DoS on the correlationbetween CAT activity and H2O2 concentration (Table 2).

At all salinity levels, APX activity was higher in Mplants than NM plants in leaves as well as roots; how-ever, significant differences between M and NM treat-ments were evident primarily in leaves (Tables 3 and 4).Compared to leaves, APX activity in roots was notablyhigher at each NaCl level at both sampling times.Three-way ANCOVA revealed a significant influence ofNaCl, AMF, DoS, and their interactions (Table 2) on thecorrelation between APX activity and H2O2 concentration(Table 2).

Table 2 Three-way ANCOVA test for independent variables NaCl, AMF,DoS and interactions between and among them on lipid peroxidation,H2O2 concentration, antioxidant molecules (ascorbic acid, α-tocopherol,glutathione, and carotenoids), activity of antioxidant enzymes (SOD, CAT,

APX, GPX, and GR), and correlations between lipid peroxidation and α-tocopherols, α-tocopherols and ascorbic acid, H2O2 and SOD, H2O2 andCAT, H2O2 and APX, and H2O2 and PX in leaves and roots of M and NMTrigonella foenum-graecum plants at 1DAT and 14DAT

Parameters Tissue NaCl AMF status DoS NaCl×AMF AMF×DoS NaCl×DoS NaCl×AMF×DoS

Lipid peroxidation Leaf *** ns *** ns ns *** ns

Root *** ns *** ns ns *** ns

Hydrogen peroxide Leaf ns ns *** ns ns ns ns

Root *** ns *** *** ns *** **

Ascorbic acid Leaf *** *** *** * ** * **

Root *** *** *** *** *** *** ***

α-Tocopherols Leaf *** * *** ns ns ** ns

Root *** ** *** ns ns * ns

Glutathione Leaf *** *** *** *** ** *** ***

Root *** *** *** ** * ** ***

Carotenoids Leaf *** *** *** ** *** *** ns

Superoxide dismutase Leaf *** ns *** * *** * ***

Root *** *** *** *** *** *** ***

Catalase Leaf ns ns ns ** ns ns ns

Root ns ns ** ns ns ns ns

Ascorbate peroxidase Leaf ns ns ns ns ns ns ns

Root *** ns ns * *** *** ***

Peroxidase Leaf ns ns *** ns ns ns ns

Root *** ns *** ns *** ns *

Glutathione reductase Leaf ns ns *** ns ns ns ns

Root ** ** *** ** ns *** **

Corr(lipid peroxidation, α-tocopherols) Leaf *** *** *** *** *** *** ***

Root *** *** *** *** *** *** ***

Corr(α-tocopherols, ascorbic acid) Leaf *** *** *** *** *** *** **

Root *** *** *** *** *** *** **

Corr(H2O2, SOD) Leaf *** *** *** *** ** *** ***

Root *** *** *** *** *** *** ***

Corr(H2O2, CAT) Leaf *** *** *** *** ns *** ***

Root *** *** *** *** *** *** ***

Corr(H2O2, APX) Leaf *** *** *** *** *** *** ***

Root *** *** *** *** *** *** *

Corr(H2O2, PX) Leaf *** *** *** *** *** *** ***

Root *** *** *** *** *** *** ***

M mycorrhizal, NM nonmycorrhizal, DAT days after treatment, DoS duration of stress, ns nonsignificant

***p <0.001; **p<0.01; *p <0.05

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Effect ofG. intraradices colonization on PX activity in leaveswas evident only during initial stages of salt stress, while it wasevident in roots at both 1DAT and 14DAT (Tables 3 and 4).

While the PX activity in leaves was not significantly influencedby the intensity of salt stress, no fixed pattern could be observedin roots. The study revealed a significant influence of NaCl,

Fig. 2 Influence of NaCl andGlomus intraradices inoculationon leaf (A–B) and root (C–D)H2O2 concentration (μg g-1 freshweight) in Trigonella foenum-graecum at 1DAT and 14DAT.Values represent mean ofreplicates; ±SD. Different lettersindicate significant differencesat p<0.05. DAT days aftertreatment, M mycorrhizal,NM nonmycorrhizal

Fig. 3 Influence of NaCl andGlomus intraradices inoculationon leaf (A–B) and root (C–D)ascorbic acid concentration (μg g-1

fresh weight) in Trigonellafoenum-graecum at 1DAT and14DAT. Values represent mean ofreplicates; ±SD. Different lettersindicate significant differences atp<0.05.DAT days after treatment,M mycorrhizal, NM non-mycorrhizal

Mycorrhiza (2014) 24:197–208 203

AMF, DoS, and their interactions (Table 2) on the correlationbetween PX activity and H2O2 concentration (Table 2).

M plants showed higher GR activity in both leaves androots than the corresponding NM plants under nonsaline as

well as saline conditions (Tables 3 and 4). GR activity in bothtissues was higher at 14DAT as compared to 1DAT. While asignificant difference between M and NM treatments wasevident in leaves at initial stages of salt stress, the effect of

Fig. 4 Influence of NaCl andGlomus intraradices inoculationon leaf (A–B) and root (C–D)α-tocopherol concentration(μg g-1 fresh weight) inTrigonella foenum-graecumat 1DAT and 14DAT. Valuesrepresent mean of replicates;±SD. Different letters indicatesignificant differences at p <0.05.DAT days after treatment, Mmycorrhizal, NM nonmycorrhizal

Fig. 5 Influence of NaCl andGlomus intraradices inoculationon leaf (A–B) and root (C–D)glutathione concentration (μg g-1

fresh weight) in Trigonellafoenum-graecum at 1DAT and14DAT. Values represent mean ofreplicates; ±SD. Different lettersindicate significant differences atp<0.05.DAT days after treatment,M mycorrhizal, NMnonmycorrhizal

204 Mycorrhiza (2014) 24:197–208

G. intraradices inoculation on GR activity in roots was sig-nificant at higher salt stress.

Discussion

Salt stress had a direct influence on G. intraradices whichresulted in a decreased ability of the fungus to colonize rootsof fenugreek. In spite of this decrease in root colonization withincreased salt stress, the mycobiont sustained benefits to thehost plant. At each level of salt stress, M plants continued toexhibit better growth than NM plants, suggesting alleviationof salt stress. The presence of excess salts has been found toreduce plant growth and biomass by inducing oxidative stressin them (Hajiboland et al. 2010; Porcel et al. 2012). Thepresent study demonstrates a decrease in NaCl-induced oxi-dative damage in fenugreek plants colonized by G.intraradices . Lower oxidative stress in M plants is due to (i)their improved growth and physiological amelioration of salt

stress tolerance by ionic and osmotic homeostasis as alreadyreported earlier (Evelin et al. 2012, 2013), and (ii) a betterantioxidative capacity of M compared to NM plants. Overall,in the present study, the activity of antioxidant enzymes (SOD,CAT, APX, PX, and GR) and concentration of antioxidantmolecules (ascorbic acid, α-tocopherol, glutathione, and ca-rotenoids) in both leaves and roots of fenugreek plants werehigher in M than NM plants. These results corroborate previ-ous observations of such higher activity of antioxidant en-zymes and concentrations of antioxidant molecules in Mplants under salt stress (ZhongQun et al. 2007; Garg andManchanda 2009; Hajiboland et al. 2010). However, theextent of effect of G. intraradices colonization on theseparameters varied with the plant tissue and the level andduration of the stress.

Salt stress causes peroxidation of membrane lipids therebydisrupting membrane integrity (Juan et al. 2005). There was asignificant effect of salt stress intensity and DoS on lipidperoxidation of fenugreek plants, indicating generation of

Fig. 6 Influence of NaCl andGlomus intraradices inoculationon leaf (A–B) carotenoidconcentration (mg g-1 fresh weight)in Trigonella foenum-graecum at1DAT and 14DAT. Valuesrepresent mean of replicates; ±SD.Different letters indicate significantdifferences at p<0.05. DAT daysafter treatment,M mycorrhizal,NM nonmycorrhizal

Table 3 Effect of NaCl andGlomus intraradices inoculation on level antioxidant enzyme activities in leaves ofM and NM Trigonella foenum-graecumplants at 1DAT and 14DAT

NaCl treatment(mM)

AM status Superoxide dismutase(nkat g-1 protein)

Catalase(μkat g-1 protein)

Ascorbate peroxidase(μkat g-1 protein)

Peroxidase(μkat g-1 protein)

Glutathione reductase(μkat g-1 protein)

DAT DAT DAT DAT DAT

1 14 1 14 1 14 1 14 1 14

0 NM 89.9a 957.3a 143.3a 236.5a 7.33a 9.8a 57.8a 6.7a 893.5a 5,945.7a

M 129.8bc 1,156a 254bc 390ab 53c 13.2ab 114.6c 13ab 1,263.5b 5,973.3a

50 NM 106.3ab 1,451.5bc 208.8ab 378.6ab 14.3a 11.1ab 54.8ab 12.7ab 1,053.7ab 6,976.7a

M 146.1cd 1,451 bc 312.8cd 451.6ab 39.6b 17.5c 113.3bc 12 ab 1,566.6c 6,898.8 a

100 NM 165.3cd 1,207.2ab 355.6cd 402.5ab 36b 14.2bc 62.2a 9.3a 1,094.3ab 8,407.2b

M 171.8d 1,696.8cd 392cd 314.5ab 44bc 28.7d 108.5bc 9.3a 1,734.8c 8,737.3b

200 NM 180.2d 1,764.7d 394.5cd 276ab 39b 16.5c 81.2ab 11.8ab 1,159.7ab 8403.7 b

M 230.5 e 2,122 e 486d 518.3b 49c 35.8 e 113.3 bc 17.7 b 1,928.3d 9,307.7b

Values within a column represent mean of replicates. Different letters within a column indicate significant differences at p <0.05

DAT days after treatment, M mycorrhizal, NM nonmycorrhizal

Mycorrhiza (2014) 24:197–208 205

more ROS when exposed to salt stress for a longer duration.However, M plants displayed lower lipid peroxidation levelsthan NM plants indicating that G. intraradices colonizationreduces oxidative damage. This observation is in accordancewith earlier reports (ZhongQun et al. 2007; Garg andManchanda 2009; Hajiboland et al. 2010) and shows thatincreased oxidative damage with increase in DoS andthe alleviation potential of AMF is more evident withincrease in salt stress severity and DoS. Prolonged ex-posure to salt stress resulted in more H2O2 productionin fenugreek plants, with concentrations increasing withincreased salt stress intensity at both time points inleaves and roots of M as well as NM plants, leading tooxidative stress. Interestingly, despite the salt stress beingapplied to roots, leaves had higher H2O2 concentration thanroots owing to higher levels of activities of the H2O2-scav-enging antioxidant enzymes, CAT, APX, and PX in rootscompared to leaves.

Under salt stress,G. intraradices colonization of fenugreekroots increased concentrations of the antioxidant moleculesα-tocopherol, ascorbic acid, glutathione, and carotenoids. Lowerlipid peroxidation in M plants was therefore due to highercontents ofα-tocopherol which disrupts the chain propagationstep in lipid auto-oxidation (Serbinonva and Packer 1994).The significant independent effects of NaCl, AMF, and DoSand their interactions on the correlation between α-tocopheroland lipid peroxidation support this conclusion. More α-tocopherol in M fenugreek plants resulted from its increasedgeneration from tocopheroxyl radicals, facilitated by thehigher concentration of ascorbic acid in them (Thomas et al.1992; Noctor and Foyer 1998). This conclusion is substanti-ated by the significant effects of NaCl, AMF, and DoS

independently and in combination on the correlation betweenα-tocopherol and ascorbic acid. Higher ascorbic acid concen-trations in M plants may, in turn, be attributed to their higherGR activity and higher concentration of glutathione (reducedform). More GSH in M than NM fenugreek plants will enablethem (i) to directly scavenge more 1O2 and H2O2 as well asother ROS-like hydroxyl radicals (Smirnoff 1993; Brivibaet al. 1998; Noctor and Foyer 1998), and (ii) to regeneratemore ascorbic acid via the ascorbate–glutathione cycle com-pared to NM plants (Foyer and Halliwell 1976, 1977). In thepresent study, the higher concentration of carotenoids in Mplants will have also contributed to the lower oxidative dam-age in them, as these molecules prevent generation of singletoxygen (Fyfe et al. 1995).

Under both saline and nonsaline conditions, M fenugreekplants showed higher activities of the antioxidant enzymesSOD, CAT, APX, PX, and GR than their NM counterparts,indicating a better ROS scavenging system in them. HigherSOD activity inM thanNMplants may be due to a stimulationof plant SOD activity or enzyme-encoding genes by the AMsymbiosis (Ruiz-Lozano et al. 2012). Besides, an isoform ofCuSO4-SOD has been reported in spores of an M fungus(Palma et al. 1993). It is worthwhile to note that SOD is ametalloenzyme and exists in different isoforms based on themetal co-factor: iron (Fe-SOD), manganese (Mn-SOD), andcopper–zinc (Cu–Zn SOD; Alscher et al. 2002). Theactivity of each SOD isoform is influenced by the avail-ability of respective co-factors (Alguacil et al. 2003). Undersalt stress, the availability of low mobility micronutrients,such as Cu, Fe, Mn, and Zn, in the soil decreases (Grattanand Grieve 1999), rendering them inaccessible for plants.In previous work, we showed that G. intraradices

Table 4 Effect of NaCl andGlomus intraradices inoculation on antioxidant enzyme activities in roots ofM and NM Trigonella foenum-graecum plantsat 1DAT and 14DAT

NaCl treatment(mM)

AMstatus

Superoxide dismutase(nkat g-1 protein)

Catalase(μkat g-1 protein)

Ascorbate peroxidase(μkat g-1 protein)

Peroxidase(μkat g-1 protein)

Glutathione reductase(μkat g-1 protein)

DAT DAT DAT DAT DAT

1 14 1 14 1 14 1 14 1 14

0 NM 510.1a 6,589a 111a 1,166bc 96ab 59a 115a 204.8a 959.2a 1,263.3a

M 714.8cd 9,137.8ab 224.7b 2,264f 102abc 82ab 205bc 179.3a 1,355.2ab 1,395ab

50 NM 574.5b 10,333.3bc 171.7ab 1,542cd 99abc 110cd 143.5a 217.8a 1,296.6abc 1,433.3ab

M 769.7d 10,487.8 bc 327.1c 1,785e 127.3c 132.1d 235.1c 341.8c 1,812.3bcd 1,895ab

100 NM 657c 12,955.6cd 198.3b 1,033bc 95a 121.8cd 159.8ab 272.2b 1,391.5abc 1,863.3ab

M 1,091.8f 18,299.9e 368.8d 1,242c 125.8bc 128d 192.1bc 331.6 c 1,973.3cd 3,836.6 c

200 NM 683.5c 14,137.8d 108a 583 a 104.3abc 97bc 145.6a 366.7 c 1,514.7 abc 2,031.6 b

M 837.8e 24,266.7f 537e 715b 115.5abc 168.6e 159.8bc 435.8d 2,135.8d 5,178.3d

Values within a column represent mean of replicates. Different letters within a column indicate significant differences at p <0.05

DAT days after treatment, M mycorrhizal, NM nonmycorrhizal

206 Mycorrhiza (2014) 24:197–208

colonization helped in better assimilation of thesemicronutrients in fenugreek plants under saline stress (Evelinet al. 2012).

Once free O2- is dismutated by SOD, there is a need to

detoxify released H2O2 by conversion to H2O in subsequentreactions. In the present study, in spite of a higher SODactivity in M fenugreek plants at each time point, the H2O2

concentration in tissues of M plants was lower than corre-sponding NM plants. This suggests that rapid scavenging ofH2O2 generated by SOD in M plants is facilitated by higheractivities of the H2O2 scavenging enzymes CAT, PX, andAPX. CAT and APX, which are metalloenzymes dependenton the availability of their co-factors. Thus, variations inresponse to DoS and NaCl can be partially explained bychanges in the availability of micronutrients to M plants undersalt stress (Evelin et al. 2012). Other than CAT, the higheractivity of antioxidative enzymes with increased salt stressmay be due to their induction by higher concentrations ofH2O2. Higher APX activity in M plants may result from moreavailability of ascorbic acid for reduction of H2O2 to H2O,which, in turn, is dependent on higher GR activity: a higherGR activity gives a higher pool of GSH, which, in turn,depletes dehydroascorbate (DHA) concentration and pro-duces more ascorbic acid (Foyer and Halliwell 1976, 1977).

At 1DAT, the CAT, APX, PX, and GR activities in leaveswere higher in M fenugreek plants than NM plants, but therewere no significant differences in the activity of these enzymesbetween M and NM plants at 14DAT, except for APX activity.These results indicate that at 1DAT, the H2O2 detoxificationsystem in leaf tissue comprises CAT, APX, PX, and GR, whileat 14DAT, APX detoxifies H2O2 generated in leaves. AlthoughM plants showed higher CAT, APX, PX, and GR activities inroots than NM plants, significant differences between M andNMplants varied with time of sampling and salt stress intensity,suggesting that the contribution of each enzyme in amelioratingoxidative stress in M plants cannot be generalized. APX andPX activities increased with increasing DoS, while althoughroot CATactivity increased over time, it declined with increasesin NaCl level. This may be explained by three factors: (i) saltstress provokes CAT degradation by endogenous proteasescausing direct functional or structural effects on CAT protein,(ii) decrease in Fe concentrations under salt stress (Shim et al.2003; Foyer and Noctor 2005), and (iii) salt stress preventssynthesis of new enzyme (Feierabend and Engel 1986).

In conclusion, AM development results in higher activitiesof antioxidant enzymes and greater production nonenzymaticantioxidant molecules. Therefore, M plants are betterequipped with an antioxidant defense system for the preven-tion of oxidative damage, and therefore they are more tolerantto salt stress. However, the combination of enzymes andantioxidant molecules used to scavenge ROS varies with theplant tissue, level of salt stress, and DoS. In the present study,the effect of G. intraradices colonization on antioxidative

enzymes in leaves and roots of enugreek plants was moreevident during initial stage of salt stress while the concentra-tion of antioxidant molecules in M plants was significantlyinfluenced with longer exposure to high salinity stress. Thepotential of AMF to ameliorate tolerance to oxidative stressgenerated by salinity was more evident at higher intensities ofsalt stress.

Acknowledgments HeikhamEvelin is grateful to theCouncil of Scientificand Industrial Research, New Delhi, India, and the University of Delhi forfinancial assistance.

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