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Research ArticleExogenous Proline and Glycine Betaine Mediated Upregulationof Antioxidant Defense and Glyoxalase Systems ProvidesBetter Protection against Salt-Induced Oxidative Stress in TwoRice (Oryza sativa L) Varieties

Mirza Hasanuzzaman1 Md Mahabub Alam2 Anisur Rahman12 Md Hasanuzzaman1

Kamrun Nahar23 and Masayuki Fujita2

1 Department of Agronomy Faculty of Agriculture Sher-e-Bangla Agricultural University Sher-e-Bangla NagarDhaka 1207 Bangladesh

2 Laboratory of Plant Stress Responses Department of Applied Biological Science Faculty of Agriculture Kagawa University2393 Ikenobe Miki-cho Kita-gun Kagawa 761-0795 Japan

3Department of Agricultural Botany Faculty of Agriculture Sher-e-Bangla Agricultural University Sher-e-Bangla NagarDhaka 1207 Bangladesh

Correspondence should be addressed to Mirza Hasanuzzaman mhzsauagyahoocom

Received 25 February 2014 Revised 9 May 2014 Accepted 9 May 2014 Published 3 June 2014

Academic Editor Qaisar Mahmood

Copyright copy 2014 Mirza Hasanuzzaman et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present study investigates the roles of exogenous proline (Pro 5mM) and glycine betaine (GB 5mM) in improving saltstress tolerance in salt sensitive (BRRI dhan49) and salt tolerant (BRRI dhan54) rice (Oryza sativa L) varieties Salt stresses(150 and 300mM NaCl for 48 h) significantly reduced leaf relative water (RWC) and chlorophyll (chl) content and increasedendogenous Pro and increased lipid peroxidation and H

2O2levels Ascorbate (AsA) glutathione (GSH) and GSHGSSG ascorbate

peroxidae (APX)monodehydroascorbate reductase (MDHAR) dehydroascorbate reductase (DHAR) glutathione reductase (GR)glutathione peroxidase (GPX) catalase (CAT) and glyoxalase I (Gly I) activities were reduced in sensitive variety and thesewere increased in tolerant variety due to salt stress The glyoxalase II (Gly II) glutathione S-transferase (GST) and superoxidedismutase (SOD) activities were increased in both cultivars by salt stress Exogenous Pro and GB application with salt stressimproved physiological parameters and reduced oxidative damage in both cultivars where BRRI dhan54 showed better toleranceThe result suggests that exogenous application of Pro and GB increased rice seedlingsrsquo tolerance to salt-induced oxidative damageby upregulating their antioxidant defense system where these protectants rendered better performance to BRRI dhan54 and Procan be considered as better protectant than GB

1 Introduction

Crop plants as sessile organisms face a number of environ-mental adversities termed as abiotic stress which includes soilsalinity water deficit extremely high or low temperaturestoxic metals waterlogging elevated ozone and ultravioletradiation which all create a barrier for proper growthmetabolism and productivity of crop plants [1ndash10] Among

the environmental stresses soil salinity is a widespread envi-ronmental problem that has been found to affect more than77million hectares or 5of the cultivable land of the universe[11 12] Salinity adversely affects the plant growth andproduc-tivity The yield reduction due to salt stress may account forsubstantial reduction of the average yield of major crops bymore than 50 [13]Thenature of damages due to salt stress isvery complex because it causes both osmotic stress and ionic

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 757219 17 pageshttpdxdoiorg1011552014757219

2 BioMed Research International

toxicity [7] At molecular level one of the common events inplants grown under salt stress is the considerable increases inreactive oxygen species (ROS) such as singlet oxygen (1O

2)

superoxide radical (O2

∙minus) hydrogen peroxide (H2O2) and

hydroxyl radical (∙OH) [2] However the production of ROSgreatly depends on the degree and duration of the impositionof stress and types of crop as well [2] Considering thedestructive effects of salt-induced oxidative stress in plants itis crucial to keep the ROS level below the toxic limit Plantsalways try to keep well-developed enzymatic and nonen-zymatic antioxidant defense system ready to encounter thedeleterious effects of ROS [2]The enzymatic system includesthe four enzymes of the ascorbate-glutathione (AsA-GSH)cycle ascorbate peroxidase (APX) monodehydroascorbatereductase (MDHAR) dehydroascorbate reductase (DHAR)and glutathione reductase (GR) as well as other enzymeslike superoxide dismutase (SOD) catalase (CAT) glutathioneperoxidase (GPX) and glutathione S-transferase (GST) Thenonenzymatic antioxidants include ascorbic acid (AsA) glu-tathione (GSH) phenolic compounds alkaloids nonproteinamino acids and 120572-tocopherols They act together in scav-enging or detoxifying ROS and subsequent protection ofplant cells from oxidative damage [2 4] However this systemacts differently in different plant species and cultivars andit was observed that the enhancement of the antioxidantdefense system is often correlated with salt stress tolerance [47] Methylglyoxal (MG) is another highly reactive cytotoxicwhich is produced largely under abiotic stress includingsalinity [14 15] and leads to damages to proteins lipids andDNA [16 17] In line with antioxidant defense system plantsalso possess glyoxalase system consisting of two enzymesglyoxalase I (Gly I) and glyoxalase II (Gly II) those candetoxify MG Enzymes of the glyoxalase system are found toregulate environmental stresses including salinity as reportedin many plant studies [2 3 10 18] It was reported that thecoordinated upregulation of both the antioxidant defense andglyoxalase systems is necessary to attain significant toleranceto oxidative stress [2 6]

It is an urgent task of plant biologists to explore suitablemechanisms of developing salt tolerant crop plants that canproduce sufficient yield under adverse condition In recentdecades many researchers have been trying to find the waysto alleviate salt stress or to overcome salt injury in plantsAmong them exogenous application of substances such asosmoprotectants phytohormones antioxidants and traceelements came to attention in recent times [4 19 20]

In response to various environmental stresses plantsdemonstrate a variety of adaptive mechanisms to counteractthem Since one of the primary responses under salt stressis osmotic adjustment compatible solutes such as proline(Pro) and glycine betaine (GB) are very common to beaccumulated during salt stress and play a fundamental rolein osmotic adjustment in plants [21] These compatiblesolutes are accumulated in the cytosol without disturbingintracellular biochemistry which ameliorate the detrimentaleffects of salinity [22ndash25]

In many plant species increased accumulation of Pro andGB were observed as an indicator of salt stress tolerance

[22 26] However most of the plants especially underelevated levels of salt cannot synthesize sufficient amountof these osmoregulators In many recent reports exogenousapplications of Pro and GB were found to act as protectantsunder salt stress [12 25 27] Besides osmoprotection Pro andGB also showed their roles in elimination of oxidative stressby triggering the antioxidant defense and also glyoxalasesystem [25 28ndash32]

Although there are several reports on the role of Pro andGB in salt stress tolerance in terms of growth and physiologyfew investigations have been done on the effects of exogenousPro and GB on both antioxidant defense and the glyoxalasesystem in rice Therefore we investigated the protectiveeffects of these osmoprotectants on the antioxidant defenseand glyoxalase systems in rice seedlings grown under salinemedia We also investigated the comparative performance oftwo modern rice varieties differing in their salt tolerance toknow their actual adaptive mechanisms under salt stress withor without osmoprotectants

2 Materials and Methods

21 Plant Materials and Stress Treatments Seeds of two rice(Oryza sativa L) cultivars cv BRRI dhan49 (salt sensitive)and cv BRRI dhan54 (salt tolerant) were collected fromBangladesh Rice Research Institute (BRRI) and surface-sterilized with 70 ethanol for 10min followed by washingseveral times with sterilized distilled water Seeds were thensoaked for 24 h in the dark Seeds were sown on plastic netsupon plastic beakers containing distilled water and kept inthe dark at 28 plusmn 2∘C for germination After 48 h uniformlygerminated seeds were transferred to growth chamber withcontrolled conditions (light intensity 100120583molmminus2 sminus1 tem-perature 25 plusmn 2∘C relative humidity 65ndash70) and duringthe growing period hyponex solution (Hyponex Japan) wasused as nutrient Two levels of salt stresses (150 and 300mMNaCl) were imposed on fourteen-day-old rice seedlings withwithout 5mM proline [l (-) Proline Wako Japan] andbetaine (Betaine Wako Japan) and where these protectantswere sprayed twice a daymixing with the wetting agent 002Tween 20 (Tween 20 Wako Japan) Control plants weregrownwithHyponex solution onlyDatawere taken after 48 hof NaCl treatment The experiment was repeated three times(119899 = 3) under the same conditions

22 Measurement of Relative Water Content Relative watercontent (RWC) was measured according to Barrs andWeath-erley [33] Leaf laminaswereweighed (freshwt FW) and thenimmediately floated on distilled water in a petri dish for 8 hin the dark Turgid weights (TW) were obtained after dryingexcess surface water with paper towels Dry weights (DW)were measured after drying at 80∘C for 48 h The calculationwas done using the following formula

RWC () = FW minus DWTW minus DW

times 100 (1)

23 Determination of Chlorophyll Content Chlorophyll con-tent was determined by homogenizing leaf samples (05 g)

BioMed Research International 3

with 10mL of acetone (80 vv) followed by centrifuging at5000timesg for 10min The absorbance was measured with aUV-visible spectrophotometer at specified wave length andchl contents were calculated using the equations proposed byArnon [34]

24 Determination of Proline Content Free proline in leaftissues was appraised following the protocol of Bates et al[35] Fresh leaf tissue (05 g) was homogenized in 10mL of 3sulfosalicylic acid in ice The homogenate was centrifuged at11500timesg for 15min Two mL of the filtrate was mixed with2mL of acid ninhydrin and 2mL of glacial acetic acid Afterincubation at 100∘C for 1 h it was cooled and 4mL of toluenewas added The optical density of the chromophore contain-ing toluene was read spectrophotometrically at 520 nm usingtoluene as a blank The amount of Pro was determined bycomparison with a standard curve

25 Measurement of Lipid Peroxidation The level of lipidperoxidation was measured by estimating MDA using thio-barbituric acid (TBA) as the reactive material following themethod of Heath and Packer [36] with slight modifica-tions The leaf samples (05 g) were homogenized in 3mL5 (wv) trichloroacetic acid (TCA) and the homogenatewas centrifuged at 11500timesg for 10min One mL supernatantwas mixed with 4mL of TBA reagent (05 of TBA in20 TCA) The reaction mixture was heated at 95∘C for30min in a water bath and then quickly cooled in an icebath and centrifuged at 11500timesg for 15min The absorbanceof the colored supernatant was measured at 532 nm andwas corrected for nonspecific absorbance at 600 nm Theconcentration ofMDAwas calculated by using the extinctioncoefficient of 155mMminus1cmminus1 and expressed as nmol of MDAgminus1 fresh weight

26 Measurement of H2O2 H2O2was assayed according to

the method described by Yu et al [37] H2O2was extracted

by homogenizing 05 g of leaf samples with 3mL of 50mMpotassium-phosphate (K-P) buffer (pH 65) at 4∘C Thehomogenate was centrifuged at 11500timesg for 15min Threemilliliter of supernatant wasmixed with 1mL of 01 TiCl

4in

20 H2SO4(vv) and kept in room temperature for 10min

After that the mixture was again centrifuged at 11500timesgfor 15min The optical absorption of the supernatant wasmeasured spectrophotometrically at 410 nm to determinethe H

2O2content (L = 028120583Mminus1cmminus1) and expressed as

nmol gminus1 fresh weight

27 Extraction and Measurement of Ascorbate and Glu-tathione Rice leaves (05 g fresh weight) were homoge-nized in 3mL ice-cold acidic extraction buffer (5 meta-phosphoric acid containing 1mM EDTA) using a mortarand pestle Homogenates were centrifuged at 11500timesg for15min at 4∘C and the supernatant was collected for analysisof ascorbate and glutathione

Ascorbate content was determined following the methodofHuang et al [38] with somemodificationsThe supernatantwas neutralized with 05M K-P buffer (pH 70) The AsA

was assayed spectrophotometrically at 265 nm in 100mM K-P buffer (pH 70) with 05 unit of ascorbate oxidase (AO) Aspecific standard curve with AsA was used for quantification

The glutathione pool was assayed according to previouslydescribed methods [37] with modifications [39] utilizing200120583L of aliquots of supernatant neutralized with 300 120583Lof 05M K-P buffer (pH 70) Based on enzymatic recy-cling GSH is oxidized by 551015840-dithio-bis (2-nitrobenzoicacid) (DTNB) and reduced by NADPH in the presenceof GR and glutathione content is evaluated by the rateof absorption changes at 412 nm of 2-nitro-5-thiobenzoicacid (NTB) generated from the reduction of DTNB GSSGwas determined after removal of GSH by 2-vinylpyridinederivatization Standard curves with known concentrationsof GSH and GSSG were used The content of GSH wascalculated by subtracting GSSG from total GSH

28 Determination of Protein The protein concentrationof each sample was determined following the method ofBradford [40] using BSA as a protein standard

29 Enzyme Extraction and Assays Using a pre-cooled mor-tar and pestle 05 g of leaf tissue was homogenized in 1mL of50mM ice-cold K-P buffer (pH 70) containing 100mM KCl1mM ascorbate 5mM 120573-mercaptoethanol and 10 (wv)glycerol The homogenates were centrifuged at 11500timesg for10min and the supernatants were used for determination ofenzyme activity All procedures were performed at 0ndash4∘C

LOX (EC 1131112) activity was estimated according tothe method of Doderer et al [41] by monitoring the increasein absorbance at 234 nm using linoleic acid as a substrateThe activity was calculated using the extinction coefficient(25mMminus1 cmminus1) and expressed as units (1 nmol of substrateoxidized per minute) per mg protein

SOD (EC 11511) activity was estimated according toBeyer and Fridovich [42] which was based on xanthine-xanthine oxidase system The reaction mixture contained K-P buffer (50mM) NBT (224mM) catalase (01 units) xan-thine oxidase (01 units) xanthine (236mM) and enzymeextract Catalase was added to avoid the H

2O2-mediated pos-

sible inactivation of CuZn-SOD SOD activity was expressedas units (amount of enzyme required to inhibit NBT reduc-tion by 50) minminus1mgminus1 protein

CAT (EC 11116) activity was measured according tothe method of Hasanuzzaman et al [2] by monitoring thedecrease of absorbance at 240 nm for 1min caused by thedecomposition of H

2O2 The reaction mixture contained

50mM K-P buffer (pH 70) 15mM H2O2and enzyme solu-

tion in a final volume of 700120583L The activity was calculatedusing the extinction coefficient of 394Mminus1cmminus1

APX (EC 111111) activity was assayed following themethod of Nakano and Asada [43] The reaction buffersolution contained 50mM K-P buffer (pH 70) 05mM AsA01mM H

2O2 01mM EDTA and enzyme extract in a final

volume of 700 120583L The activity was measured by observingthe decrease in absorbance at 290 nm for 1min using anextinction coefficient of 28mMminus1cmminus1


MDHAR (EC 1654) activity was determined by themethod ofHossain et al [44]The reactionmixture contained50mM Tris-HCl buffer (pH 75) 02mM NADPH 25mMAsA 05 unit of AO and enzyme solution in a final volumeof 700120583L The activity was calculated from the change inascorbate at 340 nm for 1min using an extinction coefficientof 62mMminus1cmminus1

DHAR (EC 1851) activity was determined by theprocedure of Nakano and Asada [43] The reaction buffercontained 50mM K-P buffer (pH 70) 25mM GSH and01mM DHAThe activity was calculated from the change inabsorbance at 265 nm for 1min using extinction coefficient of14mMminus1cmminus1

GR (EC 1642) activity was measured by the methodof Hasanuzzaman et al [3] The reaction mixture contained01M K-P buffer (pH 70) 1mM EDTA 1mMGSSG 02mMNADPH and enzyme solution in a final volume of 1mLThedecrease in absorbance at 340 nm was recorded for 1minThe activity was calculated using an extinction coefficient of62mMminus1cmminus1

GST (EC 25118) activity was determined spectropho-tometrically by the method of Hossain et al [45] withsomemodificationsThe reactionmixture contained 100mMTris-HCl buffer (pH 65) 15mM GSH 1mM 1-chloro-24-dinitrobenzene (CDNB) and enzyme solution in a finalvolume of 700 120583L The increase in absorbance was measuredat 340 nm for 1min The activity was calculated using theextinction coefficient of 96mMminus1cmminus1

GPX (EC 11119) activity was measured as describedby Elia et al [46] with slight modification using H

2O2as

a substrate The reaction mixture consisted of 100mM K-Pbuffer (pH 70) 1mM EDTA 1mMNaN

3 012mMNADPH

2mM GSH 1 unit GR 06mM H2O2 and 20120583L of sample

solution The oxidation of NADPH was recorded at 340 nmfor 1min and the activity was calculated using the extinctioncoefficient of 662mMminus1cmminus1

Glyoxalase I (EC 4415) assay was carried out accord-ing to Hasanuzzaman et al [2] Briefly the assay mixturecontained 100mM K-P buffer (pH 70) 15mM magnesiumsulfate 17mM GSH and 35mM MG in a final volume of700120583L The increase in absorbance was recorded at 240 nmfor 1min The activity was calculated using the extinctioncoefficient of 337mMminus1cmminus1

Glyoxalase II (EC 3126) activity was determinedaccording to the method of Principato et al [47] by mon-itoring the formation of GSH at 412 nm for 1min Thereactionmixture contained 100mMTris-HCl buffer (pH 72)02mM DTNB and 1mM S-d-lactoylglutathione (SLG) in afinal volume of 1mL The activity was calculated using theextinction coefficient of 136mMminus1cmminus1

210 Statistical Analysis All data obtained were subjected toanalysis of variance (ANOVA) and themean differences werecompared by a Duncanrsquos multiple range test (DMRT) usingXLSTAT v2013503 software [48] Differences at 119875 lt 005were considered significant

3 Results

Upon exposure to salt stress leaf RWCdecreased significantlyin both rice varieties when compared to their controls(Figure 1)However decline inRWCwas lower in salt tolerantcultivar BRRI dhan54 as compared to salt sensitive BRRIdhan49 At 150mM of NaCl it was decreased by 19 and12 in BRRI dhan49 and BRRI dhan54 respectively overcontrol while at 300mM NaCl the RWC decreased by 29and 28 (Figure 1) The application of Pro and GB effectivelymaintained the RWC in salt stressed seedlings In BRRIdhan49 Pro could increase RWC by 16 and 21 in seedlingsexposed to 150 and 300mM NaCl respectively while GBcould increase the RWC by 13 and 34 In case of BRRIdhan54 the increases were 10 and 20 at 150mM NaCl and6 and 34 at 300mMNaCl (Figure 1)

Leaf chl contents were decreased markedly upon expo-sure to salt stress In salt sensitive BRRI dhan49 chl 119886 contentdecreased by 21 and 31 at 150 and 300mM NaCl whilein salt tolerant BRRI dhan54 chl 119886 content decreased by6 and 20 only (Figure 2(a)) On the other hand whenthe seedlings were supplemented with exogenous Pro andGB chl 119886 content significantly increased in BRRI dhan49 atany level of salt however in BRRI dhan54 the incrementwas observed at 300mM NaCl only (Figure 2(a)) In BRRIdhan49 chl 119887 content was decreased by 21 and 31 at 150and 300mM NaCl while in BRRI dhan54 it was decreasedby 6 and 21 respectively (Figure 2(b)) Exogenous Pro andGB could maintain chl b content higher compared to theseedlings grown under salt stress However the incrementwas higher in salt sensitive BRRI dhan49 than salt tolerantBRRI dhan54 (Figure 2(b)) In both rice cultivars chl (119886 + 119887)content markedly decreased upon exposure to salt stressIn BRRI dhan49 chl (119886 + 119887) content decreased by 20 and31 compared to control while in BRRI dhan54 it decreasedby 6 and 21 (Figure 2(c)) In salt sensitive BRRI dhan49exogenous Pro and GB increased the chl (119886 + 119887) contentin the seedlings exposed to any levels of salt while in salttolerant the increase was observed in the seedlings exposedto 300mMNaCl only (Figure 2(c))

Salt stress caused a marked increase in endogenousPro content in rice seedlings of any variety however theincrement was higher at salt tolerant BRRI dhan54 com-pared to salt sensitive BRRI dhan49 In BRRI dhan49 Procontent increased by 52 and 105 at 150 and 300mM NaClrespectively (Figure 3) At the same levels of salt stressBRRI dhan54 showed 92 and 160 increase in Pro contentcompared to control Importantly exogenous Pro and GBcould also increase the endogenous Pro content furthercompared to the seedlings exposed to salt without Pro andGB supplementation However the comparative increase washigher in salt sensitive BRRI dhan49 than salt tolerant BRRIdhan54 (Figure 3)

The MDA content (indicator of lipid peroxidation)sharply increased at any level of salt stress in both ricevarieties However the rate on increment was higher in saltsensitive BRRI dhan49 In BRRI dhan49 150 and 300mMNaCl caused 76 and 159 increase in MDA content whilein BRRI dhan54 it was 41 and 95 respectively compared


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Figure 1 Leaf relative water content in salt sensitive and salttolerant rice seedlings induced by exogenous proline and glycinebetaine under salt stress S

150and S

300indicate 150mM NaCl and

300mM NaCl respectively Pro and GB indicate 5mM proline andglycine betaine spray respectively Mean (plusmnSD) was calculated fromthree replicates for each treatment Bars with different letters (smallletters for BRRI dhan49 and capital letters for BRRI dhan54) aresignificantly different at 119875 le 005 applying DMRT

to control (Figure 4(a)) The seedlings supplemented withPro and GB could maintain the level of MDA significantlylower compared to the seedlings exposed to salt stresswithoutsupplementation (Figure 4(a))

In our experiment LOX activity was sharply increasedby salt stress (Figure 4(b)) In salt sensitive BRRI dhan49the LOX activity was increased by 33 and 67 at 150 and300mM NaCl respectively compared to control while insalt tolerant BRRI dhan54 the activity was 31 and 65higher than the control However in both of the varieties theactivity of LOX was significantly declined in the salt treatedseedlings which were supplemented with exogenous Pro andGB (Figure 4(b))

The levels of H2O2also increased noticeably upon

exposure to NaCl In BRRI dhan49 the H2O2content was

increased by 35 and 69 at 150 and 300mM NaCl while inBRRI dhan54 it was increased by 32 and 63 respectivelycompared to control (Figure 4(c)) Both Pro and GB couldmaintain the H

2O2content lower in salt-stressed seedlings

compared to the seedlings grown without Pro or GB supple-mentation (Figure 4(c))

Leaf AsA content showed differential responses in tworice varieties when exposed to salt stress In salt sensitiveBRRI dhan49 salt stress at any level caused marked decreasein AsA content which was 26 and 51 lower than the control(Figure 5(a)) However in salt tolerant BRRI dhan54 AsAcontent increased by 14 at 150mM NaCl stress whileat 300mM NaCl it remained unchanged The seedlingssupplemented with exogenous Pro andGB increased the AsAcontents in the seedlings of BRRI dhan49 when exposedto salt stress at any level However in BRRI dhan54 suchprotection was observed at 300mMNaCl only (Figure 5(a))

In salt sensitive BRRI dhan49 GSH content decreased by27 and 57 in the seedlings exposed to 150 and 300mMNaCl respectively On the contrary BRRI dhan54 showedmarked increase in GSH content under salt stress which was49 and 51 higher at 150 and 300mM NaCl respectivelycompared to control (Figure 5(b)) In BRRI dhan49 theseedlings supplementedwith Pro andGB could increase GSHcontent under mild stress (150mM NaCl) while no increasewas observed in the seedlings grown under 300mM NaClHowever in BRRI dhan54 such protection (increase in GSHcontent) was observed at any level of salt stress (Figure 5(b))

The GSSG content in rice seedlings of any variety sharplyincreased at any level of salt stress In salt sensitive BRRIdhan49 the levels were increased by 143 and 224 at 150and 300mM NaCl respectively The increase of GSSG wasa bit lower in salt tolerant BRRI dhan54 which was 104 and220 higher at 150 and 300mM NaCl compared to control(Figure 5(c)) Exogenous Pro and GB on the other handmaintained the GSSG content significantly lower under saltstress compared to the seedlings grown without Pro and GBsupplementation (Figure 5(c)) However in tolerant varietyBRRI dhan54 Pro and GB did not show this protection undermild salt stress (150mM)

The ratio of GSHGSSG decreased markedly under saltstress in dose dependent manners and it greatly varied withvarieties (Figure 5(d)) In salt sensitive BRRI dhan49 150 and300mMNaCl resulted in 70 and 87 decrease in GSHGSSGration while in salt tolerant BRRI dhan54 it decreased by 21and 53 respectively compared to control (Figure 5(d))

Under salt stress the activity of SOD decreased in saltsensitive variety while it increased in salt tolerant variety(Figure 6(a)) In BRRI dhan49 the activity of SODwas 24 and29 lower than control when treated with 150 and 300mMNaCl respectively On the other hand the activity was 52 and38 higher in BRRI dhan54 at same doses of NaCl In BRRIdhan49 Pro and GB supplemented salt treated seedlingsshowed the enhancement of SOD activity However in caseof BRRI dhan54 slight increase in SOD activity was observedonly at 300mMNaCl (Figure 6(a))

Catalase activity showed differential responses in riceseedlings with variable salt tolerance levels and also inducedby salt levels (Figure 6(b)) In salt sensitive BRRI dhan49 theactivity decreased by any level of salt stress (31 and 55 lowerat 150 and 300mM NaCl resp compared to the control)Salt tolerant BRRI dhan54 showed significant increase inCAT activity under mild stress (150mM NaCl) whereas aslight decrease (11) was observed at severe stress (300mM)However exogenous Pro and GB enhanced the CAT activityin salt-treated seedlings (Figure 6(b))

Imposition of salt stress of 150mM significantly increasedthe APX activity by 40 in salt sensitive BRRI dhan49while in salt tolerant BRRI dhan54 it was increased by45 compared to control Under severe salt stress (300mMNaCl) APX activity was decreased by 27 in salt sensitivecultivar but it was increased by 27 in salt tolerant cultivar(Figure 7(a)) Exogenous Pro and GB supplementation insalt stressed seedlings maintained higher APX activitiescompared to salt stress alone whereas in salt tolerantBRRI dhan54 the activity was always higher than BRRI


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Figure 2 (a) chl 119886 (b) chl 119887 and (c) chl (119886 + 119887) content in salt sensitive and salt tolerant rice seedlings induced by exogenous proline andglycine betaine under salt stress S

150and S

300indicate 150mM NaCl and 300mM NaCl respectively Pro and GB indicate 5mM proline

and glycine betaine spray respectively Mean (plusmnSD) was calculated from three replicates for each treatment Bars with different letters (smallletters for BRRI dhan49 and capital letters for BRRI dhan54) are significantly different at 119875 le 005 applying DMRT

dhan49 (Figure 7(a)) Addition of Pro with 150mM salt stressincreasedAPX activities in BRRI dhan49 by 29 and in BRRIdhan54 it was as high as the stress condition its activities wereincreased by 46 and 17 under severe salt stress in those twocultivars (compared to salt stress alone)TheGB addition alsoincreased its activity in all the cases except for BRRI dhan54in severe stress (Figure 7(a))

Salt stress at any level decreased the MDHAR activity insalt sensitive BRRI dhan49 which were 19 and 24 lowerat 150 and 300mM NaCl respectively compared to control(Figure 7(b)) In contrast MDHAR activity in BRRI dhan54increased by 23 at 150mM NaCl while it was unaffectedunder severe salt stress (300mM NaCl) Exogenous Proand GB addition under any levels of salt stress signifi-cantly increased MDHAR activities irrespective of cultivars(Figure 7(b))

Salt stress caused a marked decrease in DHAR activityof BRRI dhan49 seedlings at any level of stress however in

salt tolerant BRRI dhan54 its activity only decreased in severestress (300mMNaCl) In BRRI dhan49 due to exogenous Proapplication DHAR activities were increased by 35 and 38 at150 and 300mM NaCl respectively In BRRI dhan54 exoge-nous Pro supplemented seedlings showed increased DHARactivities by 17 and 31 at 150 and 300mMNaCl respectivelycompared to salt stress alone (Figure 7(c)) Similarly in GBsupplemented BRRI dhan49 seedlings DHAR activities wereincreased by 35 and 31 and its activities increased by 16 and17 in BRRI dhan54 at 150 and 300mM NaCl respectivelycompared to salt stress alone (Figure 7(c))

The GR activity showed different responses in two ricevarieties in salt stress Compared to control the salt sensitiveBRRI dhan49 had decreased GR activities of 26 and 25 inexposure to 150 and 300mMNaCl respectively (Figure 7(d))In opposition salt tolerant BRRI dhan54 had significantlyhigher GR activities of 33 and 23 with 150 and 300mMNaCl respectively Nonetheless exogenous Pro and GB


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ine (120583

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gminus1

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ght)

Figure 3 Proline content in salt sensitive and salt tolerant riceseedlings induced by exogenous proline and glycine betaine undersalt stress S

150and S

300indicate 150mM NaCl and 300mM NaCl

respectively Pro and GB indicate 5mM proline and glycine betainespray respectively Mean (plusmnSD) was calculated from three replicatesfor each treatment Bars with different letters (small letters forBRRI dhan49 and capital letters for BRRI dhan54) are significantlydifferent at 119875 le 005 applying DMRT

enhanced its activity further in both sensitive and tolerantvarieties irrespective of salt doses compared to the activityin the seedlings exposed to salt stress alone (Figure 7(d))

Salt stresses of any level decreased GPX activity in bothsalt sensitive and tolerant cultivar except that mid stressincreased GPX activity in BRRI dhan54 compared to con-trol treatment (Figure 8(a)) Compared to 150 and 300mMsalt stress alone exogenous Pro and GB supplementationincreased GPX activity of BRRI dhan49 by 18 and 25respectively In BRRI dhan54 the GPX activity was unaffectedby either Pro or GB application (Figure 8(a)) But GPXactivity of both salt sensitive and tolerant cultivars wasimproved significantly by Pro or GB application under severesalt stress (300mM)

The activity of GST sharply increased in all rice seedlingsinduced by all levels of salt stress although its activity wasslightly higher in salt tolerant BRRI dhan54 (Figure 8(b))In BRRI dhan49 Pro addition with 150 and 300mM NaClresulted in 15 and 24 increases in GST activities comparedto salt stresses alone while in BRRI dhan54 its activitydid not increased further by exogenous application of ProUnder 150 and 300mM NaCl GB increased GST activity ofBRRI dhan49 by 20 and 19 respectively In BRRI dhan54exogenous GB addition did not increase GST activity under150mM NaCl treatment while it decreased under 300mMNaCl (Figure 8(b))

The activity of Gly I was different in rice varietiesdiffering in salt tolerance In salt sensitive BRRI dhan49the activity of Gly decreased by 25 and 41 upon exposureto 150 and 300mM NaCl respectively compared to control(Figure 9(a)) On the contrary salt tolerant BRRI dhan54showed 29 and 17 increase in Gly I activity when treatedwith 150 and 300mM NaCl respectively However in

both of the varieties exogenous Pro and GB enhanced theactivity of Gly I further compared to the activity in theseedlings exposed to salt with Pro and GB supplementation(Figure 9(b))

For salt sensitive BRRI dhan49 the activity of GlyII slightly increased (by 24) at 150mM NaCl while itremained unaffected at 300mM NaCl However in salttolerant BRRI dhan54 treatment with 150 and 300mMNaCl resulted in 33 and 45 increase in Gly II activity ascompared to control (Figure 9(b)) For BRRI dhan49 Proand GB supplementation could enhance the Gly II activityat any level of salt (33 and 44 at 150 and 300mM NaClresp) while in BRRI dhan54 further upregulation of Gly IIactivity was observed in seedlings grown under 150mMNaCl(Figure 9(b))

4 Discussion

Salt stress causes several biochemical and physiological alter-ations such as decrease in water content in tissues declinein photosynthetic pigments and oxidative stress Salt stressoften caused accelerated generation and reactions of ROSincluding 1O

2 O2

∙minus H2O2 and OH∙ leading to oxidative

stress [2] However components of antioxidant defense sys-tems in plants work in concert with control the cascades ofuncontrolled oxidation and protect plant cells from oxidativedamage by scavenging ROS [2 18] But in some cases suchas severe stress condition these defense systems are requiredto be upregulated more than their normal limit Compatiblesolutes like Pro and GB were found to protect the plants fromsalt-induced damages due to their role of osmoprotectionand antioxidant defense as well Several reports indicatedthat enhanced accumulations of Pro and GB are positivelycorrelated with higher tolerance to salt stress [27 28 49]

Since salt stress causes osmotic stress the decline inRWC is a common phenomenon in plants growth undersalinity and hence RWC is considered as a potent indicatorfor evaluating plants for tolerance to salt stress In ourstudy salt stress led to a significant decrease of RWC inrice leaves irrespective to NaCl concentration and the ricecultivars (Figure 1) Similar decrease in RWC due to saltstress was reported earlier [50 51] Decrease in RWC wasdue to loss of turgor that results in limited water availabilityfor cell extension processes [52] However when salt treatedseedlings were supplemented with Pro or GB they showedenhanced RWC which was due to the retention in waterin their tissue (Figure 1) The enhanced water content inplants due to exogenous application was also observed byother researchers [22 53 54] In BRRI dhan54 the RWC wasslightly higher than BRRI dhan49 which was due to its bettertolerance

In our experiment salt caused reduction in chl contentnamely chl 119886 chl 119887 and chl (119886 + 119887) in both rice varietiesHowever the reduction was higher in salt sensitive BRRIdhan49 (Figures 2(a)ndash2(c)) Salt stress often causes alterationin photosynthetic pigment biosynthesis [55] Similar decreasein chl content was observed by Amirjani [56] in riceHowever exogenous application of Pro and GB in salt treated


e

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bcb

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trol

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ro

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ro

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B


MD

A (n

mol

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fresh

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ght)

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BC B

05

1015202530354045

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trol

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S150

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ro

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B

LOX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


e

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bc b

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02468

101214161820

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

H2O2

(nm

olgminus

1fre

sh w

eigh

t)

(c)

Figure 4 (a) MDA content (b) LOX activity and (c) H2O2content in salt sensitive and salt tolerant rice seedlings induced by exogenous

proline and glycine betaine under salt stress S150

and S300

indicate 150mMNaCl and 300mMNaCl respectively Pro and GB indicate 5mMproline and glycine betaine spray respectively Mean (plusmnSD) was calculated from three replicates for each treatment Bars with different letters(small letters for BRRI dhan49 and capital letters for BRRI dhan54) are significantly different at 119875 le 005 applying DMRT

seedlings could elevate the chl content which might be dueto the higher biosynthesis of the pigmentThese results are inagreement with Raza et al [27] and Sakr et al [30]

Accumulation of Pro is often suggested as a selectioncriterion for the stress tolerance of most plant species [2224] In our experiment both rice varieties showed enhancedProaccumulation under any level of salt stress (Figure 3)However BRRI dhan54 showed comparatively higher Procontent than BRRI dhan49 due to its adaptive features ofhigher tolerance Importantly exogenous application of Proand GB in stressed plants further enhanced the endogenousPro content and in every case the effect of Pro was higherthan GB (Figure 3) More importantly in salt tolerant BRRIdhan54 the enhancement due to exogenous Pro and GB wasslightly lower than BRRI dhan49 which was probably due tothe fact that this variety has got the enhanced level of Produe to its natural tolerance capacity Pro and GB-induced

counteraction of salt stress due to enhanced Pro content wasreported earlier in different plants [27 30]

Lipid peroxidation is a well-known index for determiningthe extent of oxidative stress because increased MDA con-tent has been found to be highly correlated with oxidativedamages induced by various abiotic stresses including salinity[57] Hydrogen peroxide is a toxic compound which isinjurious to the cell and excessive accumulation of H

2O2is

one of the indicators of oxidative stress [2] In our experimentboth MDA and H

2O2were found to be increased under

salt stress which was in agreement with several previousreports [2 3 58ndash60] On the contrary salt treated seedlingssupplemented with exogenous Pro and GB showed lowerMDA and H

2O2contents ((Figures 4(a) and 4(c)) which was

due to their higher antioxidant defense system ExogenousPro and GB-induced upregulation of antioxidant defenseand concomitant decrease in MDA and H

2O2content was


a

b

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c

b

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BCA A A

C

A AB

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trol

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AsA

(nm

olgminus

1fre

sh w

eigh

t)

(a)

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ab bc

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trol

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GSH

(nm

olgminus

1fre

sh w

eigh

t)

(b)

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bc

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b bc

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C CC

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B B

0102030405060708090

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trol

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B


GSS

G (n

mol

gminus1

fresh

wei

ght)

(c)

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C

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D D

02468

10121416

Con

trol

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S150

+ P

ro

S150

+ G

B

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S300

+ P

ro

S300

+ G

B


GSH

GSS

G ra

tio

(d)

Figure 5 (a) AsA content (b) GSH content (c) GSSG content and (d) GSHGSSG ratio in salt sensitive and salt tolerant rice seedlingsinduced by exogenous proline and glycine betaine under salt stress S

150and S

300indicate 150mMNaCl and 300mMNaCl respectively Pro

and GB indicate 5mM proline and glycine betaine spray respectively Mean (plusmnSD) was calculated from three replicates for each treatmentBars with different letters (small letters for BRRI dhan49 and capital letters for BRRI dhan54) are significantly different at 119875 le 005 applyingDMRT

observed in many plant species including rice [49 61 62]In our experiment LOX activity was sharply increased insalt treated seedlings in both rice varieties (Figure 4(b)) Thishigher activity of LOX was assumed as reasons for increasedlipid peroxidation that caused oxidation of polyunsaturatedfatty acids as reported in many plant studies [63ndash65] Thisresult was correlated with higher MDA content of thoseseedlings grown under salt stress However exogenous Proand GB protected the seedlings by decreasing lipid peroxida-tion which was reflected with the lower activity of LOX [65]

AsA and GSH have vital roles in development of plantstress tolerance to adverse environmental conditions [66 67]Increased AsA or GSH content can effectively reduce ROSproduced under stress conditions including salt stress andthus prevents oxidative stress [68] In the present study weexamined the performance of salt tolerance and salt sensitiverice cultivars against different salinity levels and we also

examined how they are protected from salt stress by exoge-nous Pro orGB applicationThe result was interesting indeedBecause it was observed that under mild salt stress conditionAsA level of salt sensitive BRRI dhan49 was reduced whereasthe AsA level of BRRI dhan54 was increased (Figure 5(a))Severe salt stress also reduced the AsA level of salt sensitivecultivar and this stress maintained the AsA level of tolerantcultivar similar to the control This result is correlated toMDHAR and DHAR activities which regulate the recyclingof AsA within the cell From Figure 7 it is clear that whenthe MDHAR or DHAR activity was reduced in salt sensitiveBRRI dhan49 then its AsA levels were reduced irrespective ofdifferent salt dosesThe higherMDHAR andDHAR activitiesof salt tolerant BRRI dhan54 were also related to its AsAlevels (Figures 5(a) 7(b) and 7(c)) Exogenous Pro andGB supplementation significantly enhanced MDHAR andDHAR activities and subsequently improved AsA levels of


bc

de

ab

e

bcbc

C

A A AB

A A

0102030405060708090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


SOD

(uni

tmgminus

1pr

otei

n)

(a)

a

cb b

ed d

DBC

A AB

E

C C

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


CAT

(120583m

olm

inminus1

mgminus

1pr

otei

n)

(b)

Figure 6 Activities of SOD (a) and CAT (b) in salt sensitive and salt tolerant rice seedlings induced by exogenous proline and glycine betaineunder salt stress S

150and S

300indicate 150mMNaCl and 300mMNaCl respectively Pro and GB indicate 5mM proline and glycine betaine

spray respectively Mean (plusmnSD) was calculated from three replicates for each treatment Bars with different letters (small letters for BRRIdhan49 and capital letters for BRRI dhan54) are significantly different at 119875 le 005 applying DMRT

both cultivars under mild stress At severe stress Pro or GBenhanced the activities of two enzymes in both cultivars andtolerant cultivar AsA level was improved in all cases ButGB could not improve the AsA level of sensitive cultivarunder severe stressThe similar results regarding modulationof AsA pool and its related enzymes by exogenous Proand GB under salt stress was previously reported [28 29]They also mentioned Pro as more efficient protectant thanGB The GSHGSSG ratio is indicative of the cellular redoxbalance that plays vital roles in scavenging ROS maintainsbalance state of AsA-GSG cycle and acts as signal duringstress and numerous studies proved its protective rolesunder stress conditions [10 18 69] In the present studydrastic reduction of GSH contents and increased levels ofGSH were observed under salt stress for salt sensitive andsalt tolerant cultivars respectively (Figure 5(c)) Salt stressincreased the GSSG levels in both cultivars These combinedrendered the higher GSHGSSG ratio for tolerant cultivarBRRI dhan54 and reduced GSHGSSG in sensitive BRRIdhan49 (Figure 5(d)) Exogenous Pro and GB improved GSHand GSHGSSG ratio in tolerant cultivar in all salinity levelsBut in sensitive cultivar these parameters were only improvedby Pro under mild salt stress More efficiency of Pro ascompared to GB under salt stress was previously reported[28 29] This increased GSH content might be due to thesignificant increase in GR activities (Figure 7(d)) and higherGSHbiosynthesis [18] Previously higher reduced glutathioneGSH content and GSHGSSG ratio proved the existenceof reduced redox state in cells by exogenous Pro and GBtreatment [28 70] Previous research findings also expressedthat higher levels of GSH or GSHGSSG ratio induced byexogenous Pro and GB in turn helps to maintain the activityof GSH dependent antioxidant enzymes such as GPX GRandGST to scavengeROS fromcell [28 70 71]These findings

supported the results of our study which are mostly factualfor the salt tolerant BRRI dhan54 and in some cases for thesensitive BRRI dhan49

Salt stress-induced excess generation of ROS and sub-sequent enhanced activities of many antioxidant enzymesduring salt stress have been reported in many plant speciesImportantly the activities of antioxidant enzymes of salttolerant genotypes are upregulated under salt stress wheresalt sensitive species failed to do so [2 4 72] In ourexperiment antioxidant enzymes responded differently intwo rice varieties grown under two levels of salt stressExogenous application of Pro and GB also showed theirprotective effect differently in those varieties Superoxidedismutase is the first line of antioxidant enzyme that removesO2

∙minus by catalyzing its dismutation one O2

∙minus being reducedto H2O2and another oxidized to O

2[4] The enhanced

activity of SODs minimizes abiotic oxidative stress and hasa significant role in the adaptation of a plant to stressed envi-ronments [4] In our experiment salt stress downregulatedthe SOD activity in salt sensitive BRRI dhan49 while it clearlyupregulated in salt tolerant BRRI dhan54 However Proand GB supplementation could enhance the activity furtherwhich indicated its role in ROS detoxification (Figure 6(a))Catalase is a potential enzyme which has higher turnoverrate and is capable to dismutase two molecules of H

2O2to

water and oxygen and thus is considered as an efficient ROSdetoxifier [4] There are plenty of reports on the changes inCAT activity or expression and those supported the notionthat CAT is the most efficient H

2O2scavenging enzyme [4

73] In our experiment salt sensitive BRRI dhan49 showeddecline in CAT activity at any level of NaCl which might bedue to ineffective enzyme synthesis or change in assemblyof enzyme subunits (Figure 6(b)) [74 75] On the contraryin salt tolerant BRRI dhan54 the activity slightly increased


c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

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S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)

Figure 7 Activities of APX (a) MDHAR (b) DHAR (c) and GR (d) in salt sensitive and salt tolerant rice seedlings induced by exogenousproline and glycine betaine under salt stress S

150and S

300indicate 150mMNaCl and 300mMNaCl respectively Pro and GB indicate 5mM

proline and glycine betaine spray respectively Mean (plusmnSD) was calculated from three replicates for each treatment Bars with different letters(small letters for BRRI dhan49 and capital letters for BRRI dhan54) are significantly different at 119875 le 005 applying DMRT

under mild salt stress (150mM NaCl) but decreased undersevere stress (300mM NaCl) This trend was supported byearlier reports [58 76 77]However the activity of CAT in thepresence of Pro andGBunder salt treatmentwasmuchhigherthan those under salt treatment without Pro and GB whichsuggest a unambiguous role of Pro and GB in scavengingH2O2under salt stress Similar effects were also observed in

several recent studies [28 31 49 78]The four enzymes of AsA-GSH namely APX MDHAR

DHAR and GR are vital for antioxidant defense becausethey are involved in maintaining the AsA and GSH pool Inour experiment APX activity increased in both rice varietiessubjected to salt stress and exogenous Pro and GB enhancedthe activity further which indicated theH

2O2scavenging role

of Pro and GB (Figure 7(a)) This result was in agreementwith other findings [6 25 49] MDHAR and DHAR aretwo important enzymes related to the regeneration of AsAwhich is a strong antioxidant Both enzymes are equally

important in regulating AsA level and its redox state underoxidative stress condition [79ndash81] The activity of MDHARand DHAR clearly decreased in salt sensitive BRRI dhan49treated with NaCl However in salt tolerant BRRI dhan54the activity increased or remained unchanged dependingon the salt concentration (Figures 7(b) and 7(c)) Decreaseof MDHAR and DHAR activity under salt stress was alsoreported in our earlier studies [2 3] Exogenous Pro and GBsupplemented seedlings on the other hand enhanced theactivity in both varieties which helped the plants in efficientregeneration of AsA (Figure 5(a)) Glutathione reductaseis another important enzyme of AsA-GSH cycle which isimportant for maintaining high ratio of GSHGSSG in plantcells also necessary for accelerating the H

2O2scavenging

[82 83] In salt sensitive BRRI dhan49 the activity of GRdeclined under salt stress in dose dependent manners butin salt tolerant BRRI dhan54 it declined only at 300mMNaCl (Figure 7(d)) Higher activity of GR in stress tolerant


a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

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B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)

Figure 8 Activities of GPX (a) and GST (b) in salt sensitive and salt tolerant rice seedlings induced by exogenous proline and glycine betaineunder salt stress S

150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)

Figure 9 Activities of Gly I (a) and Gly II (b) in salt sensitive and salt tolerant rice seedlings induced by exogenous proline and glycinebetaine under salt stress S

150and S

300indicate 150mMNaCl and 300mMNaCl respectively Pro and GB indicate 5mM proline and glycine

betaine spray respectively Mean (plusmnSD) was calculated from three replicates for each treatment Bars with different letters (small letters forBRRI dhan49 and capital letters for BRRI dhan54) are significantly different at 119875 le 005 applying DMRT

plants was observed in several studies [76 84] However inboth rice varieties supplementation of Pro and GB in salttreated seedlings showed enhanced activities of GR whichcouldmaintain a high GSH pool (Figures 5(b) and 7(d)) Ourresults were corroborated with other recent findings whereexogenous Pro andGB upregulated the GR activity under saltstress [15 25 28]

The GPX is another vital enzyme of antioxidant defensesystem and due to substrate specifications and strongeraffinity for H

2O2it can efficiently scavenge especially H

2O2

and thus provide protection against stress [4 85] In our

experiment salt tolerant variety showed higher activities ofGPX compared to salt sensitive variety which was due to anincreased synthesis of the enzymes or an increased activationof constitutive enzyme pools (Figure 8(a)) In salt sensitivevariety GPX activity declined at any level of salt stress Thisindicates inefficient detoxification of ROS in salt sensitivevariety (BRRI dhan49) Differential response of GPX activityin salt sensitive and tolerant varieties was reported in manyplant studies [2 3 86] However in salt sensitive varietyexogenous Pro and GB upregulated the activity of GR atany level of stress In contrary in salt tolerant variety (BRRI


dhan54) Pro and GB supplemented seedlings showed furtherincrease in GR activity only at severe stress which indicatesthat under mild stress the activity was well enough to scav-enge the excess H

2O2(Figures 8(a) and 4(c)) It is interesting

thatGST activity increased sharply in both of the rice varietiesexposed to NaCl Although the primary role of this enzymehas been assigned to the detoxification of xenobiotics it hasalso been shown to exhibit antioxidant activity [4] PlantGSTs are also associated with responses to various forms ofabiotic stress [45 87] and stress tolerance is often correlatedwith enhanced activity of GST [4] In both rice varietiesof our experiment GST activity markedly increased undersalt stress where comparatively higher activity was observedin salt tolerant BRRI dhan54 (Figure 8(b)) Interestinglyexogenous Pro and GB only resulted in further enhancementof GST activity in BRRI dhan49 only which indicates that thesalt tolerant BRRI dhan54 has already got enough capacityto detoxify H

2O2(Figures 8(b) and 4(c)) Our results are

partially supported by Hoque et al [29]Glyoxalase system consisting of Gly I and Gly II enzymes

effectively detoxifyMG in two-step reactions where GSH actsas a cofactor In first step Gly I catalyzes the formation of S-d-lactoylglutathione (SLG) from the hemithioacetal formednonenzymatically from MG and GSH In second step Gly IIcatalyzes the hydrolysis of SLG to regenerate GSH and released-lactate [17 88] Effective glyoxalase system converts MGto d-lactate and at the same time it recycles GSH properly[2 3] Several studies proved that efficient glyoxalase systemnot only detoxifies MG but also improves stress tolerance[10 18] Different levels of salt stresses significantly reducedGly I activities in salt sensitive BRRI dhan49 Decrease inGly I activity under salt stress was also reported previously[2 89]On the contrary those stresses enhancingGly I activityin salt tolerant BRRI dhan54 cultivar might be an indicationof its better tolerance capacity Moreover exogenous Proand GB supplementation with salt media improving GlyI activity in two cultivars in different stress conditionsproves the enhanced tolerance level induced by Pro andGB (Figure 9(a)) After salt exposure Gly II activity of saltsensitive BRRI dhan49 was increased only under mild saltstress On the other hand tolerant BRRI dhan54 increasedits activity in both stress levels (Figure 9(b)) Enhanced GlyII activity in response to salt stress was also observed in ourprevious study [90] When these cultivars were supplied withexogenous Pro or GB with salt stress their Gly II activitiessignificantly enhanced Pro- and GB-induced upregulationof glyoxalase enzymes and subsequent salt stress tolerancewere also reported by other researchers [29 32] Engineeringof glyoxalase pathway enzymes in different plant speciesreduced MG content under salt that was correlated to GSH-based MG detoxification system which reduced oxidativedamage [16 17 91ndash93] These results are in line with ourfindings If the glyoxalase enzymes of two cultivars arecompared it can be said that the tolerant BRRI dhan54 hadhigher Gly I and Gly II activities in all the cases comparedto BRRI dhan49 Even if the GSH level of these two cultivarsare compared it is clear that BRRI dhan54 is better performeras it maintained higher GSH levels both under salt stress

conditions with or without Pro and GB (Figure 5(b)) Thehigher GSH level with higher Gly I and Gly II activities areevidence for efficient glyoxalase system for conferring saltstress tolerance [18 90]Thus higher Gly I andGly II activitieswith higher GSH level induced by Pro and GB have madeBRRI dhan54 more salt tolerant cultivar than BRRI dhan49

5 Conclusion

Considering the above results we therefore conclude thatexogenous Pro and GB are effective protectants to improveshort-term salt tolerance both in salt sensitive BRRI dhan49and salt tolerant BRRI dhan54 as Pro and GB effectivelymaintained better physiological conditions and significantlyalleviated oxidative damages of rice seedlings by enhancingthe antioxidant and glyoxalase systems In all the casesBRRI dhan54 was a better performer under salt stressAlthough the studied antioxidant and glyoxalase enzymeswere improved by both the osmoprotectants the GB couldnot restore the nonenzymatic antioxidants of salt sensitivecultivar BRRI dhan49 in some cases Considering these factsit can be assumed that Pro is good for salt sensitive cultivarat present studied condition From the comparative studiesof salt tolerant and sensitive cultivars it can be said thatenhancement of tolerance by Pro and GB even in the salt sen-sitive cultivar is an interesting point and this result deservesfurther intrinsic researches The dose duration dependentstudy with Pro and GB in different salinity levels might beelucidated Mechanisms of Pro and GB as osmoprotectantsfree radical scavengers enzyme activators or as regulators ofother physiological processes were stated inmany articles butmerely studied under different stress conditions Studies ontheir protective mechanisms and signaling cascades are thefurther scope of research

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was partially supported by Sher-e-Bangla Agri-cultural University Research System (SAURES) Grant noSAUSAURES1338 to Mirza Hasanuzzaman The authorsthank Dr MD Motiar Rohman Molecular Plant Breed-ing Laboratory Bangladesh Agricultural Research InstituteGazipur for his kind help in measuring enzymatic activ-ities The authors would like to thank Mrs Ratna RaniMajumder Genetic resource and Seed Division BangladeshRice Research Institute Gazipur for generously providingquality seeds of rice

References

[1] M Hasanuzzaman M Fujita M N Islam K U Ahamedand K Nahar ldquoPerformance of four irrigated rice varieties


under different levels of salinity stressrdquo International Journal ofIntegrative Biology vol 6 pp 85ndash90 2009

[2] M Hasanuzzaman M A Hossain and M Fujita ldquoNitricoxide modulates antioxidant defense and the methylglyoxaldetoxification system and reduces salinity-induced damage ofwheat seedlingsrdquo Plant Biotechnology Reports vol 5 no 4 pp353ndash365 2011

[3] M Hasanuzzaman M A Hossain and M Fujita ldquoSelenium-induced up-regulation of the antioxidant defense and methyl-glyoxal detoxification system reduces salinity-induced damagein rapeseed seedlingsrdquo Biological Trace Element Research vol143 no 3 pp 1704ndash1721 2011

[4] M Hasanuzzaman M A Hossain J A Teixeira da Silva andM Fujita ldquoPlant responses and tolerance to abiotic oxidativestress antioxidant defense is a key factorrdquo in Crop Stress andIts Management Perspectives and Strategies V Bandi A KShanker C Shanker and M Mandapaka Eds pp 261ndash316Springer Berlin Germany 2012

[5] M Hasanuzzaman M A Hossain and M Fujita ldquoExoge-nous selenium pretreatment protects rapeseed seedlings fromcadmium-induced oxidative stress by upregulating antioxidantdefense and methylglyoxal detoxification systemsrdquo BiologicalTrace Element Research vol 149 no 2 pp 248ndash261 2012

[6] M Hasanuzzaman K Nahar M Alam and M Fujita ldquoExoge-nous nitric oxide alleviates high temperature induced oxidativestress in wheat (Triticum aestivum) seedlings bymodulating theantioxidant defense and glyoxalase systemrdquo Australian Journalof Crop Science vol 6 no 8 pp 1314ndash1323 2012

[7] M Hasanuzzaman K Nahar and M Fujita ldquoPlant response tosalt stress and role of exogenous protectants to mitigate salt-induced damagesrdquo in Ecophysiology and Responses of Plantsunder Salt Stress P Ahmad M M Azooz and M N V PrasadEds pp 25ndash87 Springer New York NY USA 2013

[8] M Hasanuzzaman K Nahar andM Fujita ldquoExtreme tempera-tures oxidative stress and antioxidant defense in plantsrdquo inAbi-otic StressmdashPlant Responses and Applications in Agriculture KVahdati and C Leslie Eds pp 169ndash205 InTech RijekaCroatia2013

[9] M Hasanuzzaman K Nahar S S Gill andM Fujita ldquoDroughtstress responses in plants oxidative stress and antioxidantdefenserdquo in Climate Change and Plant Abiotic Stress ToleranceS S Gill and N Tuteja Eds pp 209ndash249 Wiley WeinheimGermany 2014

[10] M Hasanuzzaman and M Fujita ldquoExogenous sodium nitro-prusside alleviates arsenic-induced oxidative stress in wheat(Triticum aestivum L) seedlings by enhancing antioxidantdefense and glyoxalase systemrdquo Ecotoxicology vol 22 no 3 pp584ndash596 2013

[11] W Wang B Vinocur O Shoseyour and A Altman ldquoBiotech-nology of plant osmotic stress tolerance physiological andmolecular considerationsrdquo Acta Horticulturae vol 590 pp286ndash292 2001

[12] H R Athar and M Ashraf ldquoStrategies for crop improvementagainst salt and water stress an overviewrdquo in Salinity andWaterStress Improving Crop Efficiency M Ashraf M Ozturk and HR Athar Eds pp 1ndash16 Springer Dordrecht The Netherlands2009

[13] E A Bray J Bailey-Serres and E Weretilnyk ldquoResponses toabiotic stressrdquo in Biochemistry and Molecular Biology of PlantsB B Buchanan W Gruissem and R Jones Eds pp 1158ndash1203American Society of Plant Physiologists Rockville Maryland2000

[14] S K Yadav S L Singla-Pareek and S K Sopory ldquoAn overviewon the role of methylglyoxal and glyoxalases in plantsrdquo DrugMetabolism and Drug Interactions vol 23 no 1-2 pp 51ndash682008

[15] V Kumar and S K Yadav ldquoProline and betaine provide pro-tection to antioxidant andmethylglyoxal detoxification systemsduring cold stress in Camellia sinensis (L) O Kuntzerdquo ActaPhysiologiae Plantarum vol 31 no 2 pp 261ndash269 2009

[16] S K Yadav S L Singla-Pareek M Ray M K Reddy and SK Sopory ldquoMethylglyoxal levels in plants under salinity stressare dependent on glyoxalase I and glutathionerdquoBiochemical andBiophysical Research Communications vol 337 no 1 pp 61ndash672005

[17] S K Yadav S L Singla-Pareek M K Reddy and S K SoporyldquoTransgenic tobacco plants overexpressing glyoxalase enzymesresist an increase inmethylglyoxal andmaintain higher reducedglutathione levels under salinity stressrdquo FEBS Letters vol 579no 27 pp 6265ndash6271 2005

[18] M Hasanuzzaman and M Fujita ldquoSelenium pretreatmentupregulates the antioxidant defense andmethylglyoxal detoxifi-cation system and confers enhanced tolerance to drought stressin rapeseed seedlingsrdquo Biological Trace Element Research vol143 no 3 pp 1758ndash1776 2011

[19] M L Lopez and S M E Satti ldquoCalcium and potassiumenhanced growth and yield of tomato under sodium chloridestressrdquo Plant Sciience vol 114 no 1 pp 19ndash27 1996

[20] S Noreen H U R Athar and M Ashraf ldquoInteractive effectsof watering regimes and exogenously applied osmoprotectantson earliness indices and leaf area index in cotton (Gossypiumhirsutum L) croprdquo Pakistan Journal of Botany vol 45 pp 1873ndash1881 2013

[21] L Szabados and A Savoure ldquoProline a multifunctional aminoacidrdquo Trends in Plant Science vol 15 no 2 pp 89ndash97 2010

[22] M Ashraf and M R Foolad ldquoRoles of glycine betaine and pro-line in improving plant abiotic stress resistancerdquo Environmentaland Experimental Botany vol 59 no 2 pp 206ndash216 2007

[23] T H H Chen and N Murata ldquoGlycinebetaine an effectiveprotectant against abiotic stress in plantsrdquo Trends in PlantScience vol 13 no 9 pp 499ndash505 2008

[24] S Hayat Q Hayat M N Alyemeni A S Wani J Pichtel andA Ahmad ldquoRole of proline under changing environments areviewrdquo Plant Signaling and Behavior vol 7 no 11 pp 1ndash11 2012

[25] V Y Patade V H Lokhande and P Suprasanna ldquoExogenousapplication of proline alleviates salt induced oxidative stressmore efficiently than glycine betaine in sugarcane culturedcellsrdquo Sugar Tech vol 16 no 1 pp 22ndash29 2014

[26] M Ashraf and P J C Harris ldquoPotential biochemical indicatorsof salinity tolerance in plantsrdquo Plant Science vol 166 no 1 pp3ndash16 2004

[27] S H Raza H U R Athar and M Ashraf ldquoInfluence of exoge-nously applied glycinebetaine on the photosynthetic capacityof two differently adapted wheat cultivars under salt stressrdquoPakistan Journal of Botany vol 38 no 2 pp 241ndash251 2006

[28] M A Hoque M N A Banu E Okuma et al ldquoExogenousproline and glycinebetaine increase NaCl-induced ascorbate-glutathione cycle enzyme activities and proline improves salttolerance more than glycinebetaine in tobacco Bright Yellow-2suspension-cultured cellsrdquo Journal of Plant Physiology vol 164no 11 pp 1457ndash1468 2007

[29] M A Hoque M N A Banu Y Nakamura Y Shimoishi andY Murata ldquoProline and glycinebetaine enhance antioxidantdefense and methylglyoxal detoxification systems and reduce


NaCl-induced damage in cultured tobacco cellsrdquo Journal ofPlant Physiology vol 165 no 8 pp 813ndash824 2008

[30] M T Sakr NM El-Sarkassy andM P Fuller ldquoOsmoregulatorsproline and glycine betaine counteract salinity stress in canolardquoAgronomy for Sustainable Development vol 32 no 3 pp 747ndash754 2012

[31] K Nawaz and M Ashraf ldquoExogenous application of glycine-betaine modulates activities of antioxidants in maize plantssubjected to salt stressrdquo Journal of Agronomy and Crop Sciencevol 196 no 1 pp 28ndash37 2010

[32] M N A Banu M A Hoque M Watanabe-Sugimoto etal ldquoProline and glycinebetaine ameliorated NaCl stress viascavenging of hydrogen peroxide and methylglyoxal but notsuperoxide or nitric oxide in tobacco cultured cellsrdquo BioscienceBiotechnology and Biochemistry vol 74 no 10 pp 2043ndash20492010

[33] H D Barrs and P E Weatherley ldquoA re-examination of therelative turgidity technique for estimating water deficits inleavesrdquo Australian Journal of Biological Science vol 15 pp 413ndash428 1962

[34] D T Arnon ldquoCopper enzymes in isolated chloroplastspolyphenaloxidase in Beta vulgarisrdquo Plant Physiology vol 24pp 1ndash15 1949

[35] L S Bates R PWaldren and I D Teare ldquoRapid determinationof free proline for water-stress studiesrdquo Plant and Soil vol 39no 1 pp 205ndash207 1973

[36] R L Heath and L Packer ldquoPhotoperoxidation in isolatedchloroplasts I Kinetics and stoichiometry of fatty acid peroxi-dationrdquo Archives of Biochemistry and Biophysics vol 125 no 1pp 189ndash198 1968

[37] C W Yu T M Murphy and C H Lin ldquoHydrogen peroxide-induced chilling tolerance in mung beans mediated throughABA-independent glutathione accumulationrdquo Functional PlantBiology vol 30 no 9 pp 955ndash963 2003

[38] C Huang W He J Guo X Chang P Su and L ZhangldquoIncreased sensitivity to salt stress in an ascorbate-deficientArabidopsismutantrdquo Journal of Experimental Botany vol 56 no422 pp 3041ndash3049 2005

[39] A Paradiso R Berardino M C de Pinto et al ldquoIncreasein ascorbate-glutathione metabolism as local and precocioussystemic responses induced by cadmium in durum wheatplantsrdquo Plant and Cell Physiology vol 49 no 3 pp 362ndash3742008

[40] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[41] A Doderer I Kokkelink S van der Veen B E Valk A WSchram and A C Douma ldquoPurification and characterizationof two lipoxygenase isoenzymes from germinating barleyrdquoBiochimica et Biophysica Acta vol 1120 no 1 pp 97ndash104 1992

[42] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[43] Y Nakano and K Asada ldquoHydrogen peroxide is scavengedby ascorbate-specific peroxidase in spinach chloroplastsrdquo Plantand Cell Physiology vol 22 no 5 pp 867ndash880 1981

[44] M A Hossain Y Nakano and K Asada ldquoMonodehydroascor-bate reductase in spinach chloroplasts and its participation inregeneration of ascorbate for scavenging hydrogen peroxiderdquoPlant and Cell Physiology vol 25 no 3 pp 385ndash395 1984

[45] M Z Hossain M D Hossain and M Fujita ldquoInduction ofpumpkin glutathione S-transferases by different stresses and itspossible mechanismsrdquo Biologia Plantarum vol 50 no 2 pp210ndash218 2006

[46] A C Elia R Galarini M I Taticchi A J M Dorr andL Mantilacci ldquoAntioxidant responses and bioaccumulation inIctalurus melas under mercury exposurerdquo Ecotoxicology andEnvironmental Safety vol 55 no 2 pp 162ndash167 2003

[47] G B Principato G Rosi and V Talesa ldquoPurification andcharacterization of two forms of glyoxalase II from the liver andbrain of Wistar ratsrdquo Biochimica et Biophysica Acta vol 911 no3 pp 349ndash355 1987

[48] AddinsoftXLSTAT 2013 v2013603 Data analysis and statisticssoftware for Microsoft Excel Addinsoft Paris France 2013

[49] N Nounjan P T Nghia and P Theerakulpisut ldquoExogenousproline and trehalose promote recovery of rice seedlings fromsalt-stress and differentially modulate antioxidant enzymes andexpression of related genesrdquo Journal of Plant Physiology vol 169no 6 pp 596ndash604 2012

[50] L Vysotskaya P E Hedley G Sharipova et al ldquoEffect ofsalinity on water relations of wild barley plants differing in salttolerancerdquo AoB Plant vol 2010 p plq006 2010

[51] N Chaparzadeh and F Mehrnejad ldquoOxidative markers in fiveIranian alfalfa (Medicago sativa L) cultivars under salinitystressrdquo Iranian Journal of Plant Physiology vol 3 pp 793ndash7992013

[52] N Katerji J W van Hoorn A Hamdy M Mastrorilli and EMoukarzel ldquoOsmotic adjustment of sugar beets in response tosoil salinity and its influence on stomatal conductance growthand yieldrdquo Agricultural Water Management vol 34 no 1 pp57ndash69 1997

[53] K Nawaz and M Ashraf ldquoImprovement in salt tolerance ofmaize by exogenous application of glycinebetaine growth andwater relationsrdquo Pakistan Journal of Botany vol 39 no 5 pp1647ndash1653 2007

[54] H M A El-Samad M A K Shaddad and N BarakatldquoImprovement of plants salt tolerance by exogenous applicationof amino acidsrdquo Journal of Medicinal Plant Research vol 5 no24 pp 5692ndash5699 2011

[55] K Maxwell and G N Johnson ldquoChlorophyll fluorescencemdashapractical guiderdquo Journal of Experimental Botany vol 51 no 345pp 659ndash668 2000

[56] M R Amirjani ldquoEffect of salinity stress on growth sugarcontent pigments and enzyme activity of ricerdquo InternationalJournal of Botany vol 7 no 1 pp 73ndash81 2011

[57] N Garg and G Manchanda ldquoROS generation in plants boonor banerdquo Plant Biosystems vol 143 no 1 pp 81ndash96 2009

[58] MM Azooz AM Ismail andM F A Elhamd ldquoGrowth lipidperoxidation and antioxidant enzyme activities as a selectioncriterion for the salt tolerance of maize cultivars grown undersalinity stressrdquo International Journal of Agriculture and Biologyvol 11 no 1 pp 21ndash26 2009

[59] P Saha P Chatterjee and A K Biswas ldquoNaCl pretreatmentalleviates salt stress by enhancement of antioxidant defensesystemandosmolyte accumulation inmungbean (Vigna radiataLWilczek)rdquo Indian Journal of Experimental Biology vol 48 no6 pp 593ndash600 2010

[60] W Weisany Y Sohrabi G Heidari A Siosemardeh and KGhassemi-Golezani ldquoChanges in antioxidant enzymes activityand plant performance by salinity stress and zinc application insoybean (Glycine max L)rdquo Plant Omics Journal vol 5 no 2 pp60ndash67 2012


[61] Z Yan S Guo S Shu J Sun and T Tezuka ldquoEffects ofproline on photosynthesis root reactive oxygen species (ROS)metabolism in two melon cultivars (Cucumis melo L) underNaCl stressrdquoAfrican Journal of Biotechnology vol 10 no 80 pp18381ndash18390 2011

[62] NNounjan andPTheerakulpisut ldquoEffects of exogenous prolineand trehalose on physiological responses in rice seedlings dur-ing salt-stress and after recoveryrdquo Plant Soil and Environmentvol 58 no 7 pp 309ndash315 2012

[63] A Aziz and F Larher ldquoOsmotic stress induced changes in lipidcomposition and peroxidation in leaf discs of Brassica napus LrdquoJournal of Plant Physiology vol 153 no 5-6 pp 754ndash762 1998

[64] A Molassiotis T Sotiropoulos G Tanou G Diamantidis andI Therios ldquoBoron-induced oxidative damage and antioxidantand nucleolytic responses in shoot tips culture of the applerootstock EM 9 (Malus domestica Borkh)rdquo Environmental andExperimental Botany vol 56 no 1 pp 54ndash62 2006

[65] E Sanchez-Rodrıguez M D M Rubio-Wilhelmi B BlascoR Leyva L Romero and J M Ruiz ldquoAntioxidant responseresides in the shoot in reciprocal grafts of drought-tolerant anddrought-sensitive cultivars in tomato under water stressrdquo PlantScience vol 188-189 pp 89ndash96 2012

[66] G M Pastori G Kiddle J Antoniw et al ldquoLeaf vitamin Ccontents modulate plant defense transcripts and regulate genesthat control development through hormone signalingrdquo ThePlant Cell vol 15 no 4 pp 939ndash951 2003

[67] L Ball G-P Accotto U Bechtold et al ldquoEvidence for a directlink between glutathione biosynthesis and stress defense geneexpression in Arabidopsisrdquo The Plant Cell vol 16 no 9 pp2448ndash2462 2004

[68] A Shalata and P M Neumann ldquoExogenous ascorbic acid(vitamin C) increases resistance to salt stress and reduces lipidperoxidationrdquo Journal of Experimental Botany vol 52 no 364pp 2207ndash2211 2001

[69] J M Li and H Jin ldquoRegulation of brassinosteroid signalingrdquoTrends in Plant Science vol 12 no 1 pp 37ndash41 2007

[70] N Kumar M Pal A Singh R K SaiRam and G C SrivastavaldquoExogenous proline alleviates oxidative stress and increasevase life in rose (Rosa hybrida L rsquoGrand Galarsquo)rdquo ScientiaHorticulturae vol 127 no 1 pp 79ndash85 2010

[71] P Ghezzi V Bonetto and M Fratelli ldquoThiol-disulfide balancefrom the concept of oxidative stress to that of redox regulationrdquoAntioxidants and Redox Signaling vol 7 no 7-8 pp 964ndash9722005

[72] N Ghosh M K Adak P D Ghosh S Gupta D N S Guptaand C Mandal ldquoDifferential responses of two rice varieties tosalt stressrdquo Plant Biotechnology Reports vol 5 no 1 pp 89ndash1032011

[73] A Mhamdi G Queval S Chaouch S Vanderauwera F vanBreusegem and G Noctor ldquoCatalase function in plants a focuson Arabidopsis mutants as stress-mimic modelsrdquo Journal ofExperimental Botany vol 61 no 15 pp 4197ndash4220 2010

[74] M Gupta P Sharma N B Sarin and A K Sinha ldquoDifferentialresponse of arsenic stress in two varieties of Brassica juncea LrdquoChemosphere vol 74 no 9 pp 1201ndash1208 2009

[75] I Khan A Ahmad and M Iqbal ldquoModulation of antioxidantdefence system for arsenic detoxification in Indian mustardrdquoEcotoxicology and Environmental Safety vol 72 no 2 pp 626ndash634 2009

[76] A H Sekmen I Turkan and S Takio ldquoDifferential responsesof antioxidative enzymes and lipid peroxidation to salt stressin salt-tolerant Plantago maritima and salt-sensitive Plantagomediardquo Physiologia Plantarum vol 131 no 3 pp 399ndash411 2007

[77] C-J Lin C-Y Li S-K Lin et al ldquoInfluence of high temperatureduring grain filling on the accumulation of storage proteins andgrain quality in rice (Oryza sativa L)rdquo Journal of Agriculturaland Food Chemistry vol 58 no 19 pp 10545ndash10552 2010

[78] C Ben Ahmed B Ben Rouina S Sensoy M Boukhriss andF Ben Abdullah ldquoExogenous proline effects on photosyntheticperformance and antioxidant defense system of young olivetreerdquo Journal of Agricultural and Food Chemistry vol 58 no 7pp 4216ndash4222 2010

[79] A E Eltayeb N Kawano G H Badawi et al ldquoEnhancedtolerance to ozone and drought stresses in transgenic tobaccooverexpressing dehydroascorbate reductase in cytosolrdquo Physi-ologia Plantarum vol 127 no 1 pp 57ndash65 2006

[80] A E Eltayeb N Kawano G H Badawi et al ldquoOverexpressionofmonodehydroascorbate reductase in transgenic tobacco con-fers enhanced tolerance to ozone salt and polyethylene glycolstressesrdquo Planta vol 225 no 5 pp 1255ndash1264 2007

[81] Z Wang Y Xiao W Chen K Tang and L Zhang ldquoIncreasedvitamin C content accompanied by an enhanced recyclingpathway confers oxidative stress tolerance in ArabidopsisrdquoJournal of Integrative Plant Biology vol 52 no 4 pp 400ndash4092010

[82] A S V Chalapathi Rao and A R Reddy ldquoGlutathione reduc-tase a putative redox regulatory system in the plant cellsrdquo inSulfur Assimilation and Abiotic Stresses in Plants N A Khan SSingh and S Umar Eds pp 111ndash147 Springer Berlin Germany2008

[83] C H Pang and B S Wang ldquoRole of ascorbate peroxidaseand glutathione reductase in ascorbate-glutathione cycle andstress tolerance in plantsrdquo in Ascorbate-Glutathione Pathwayand Stress Tolerance in Plants N A Anjum M T Chan and SUmar Eds pp 91ndash112 Springer Dordrecht The Netherlands2010

[84] K Aghaei A A Ehsanpour and S Komatsu ldquoPotato respondsto salt stress by increased activity of antioxidant enzymesrdquoJournal of Integrative Plant Biology vol 51 no 12 pp 1095ndash11032009

[85] R Brigelius-Flohe and L Flohe ldquoIs there a role of glutathioneperoxidases in signaling and differentiationrdquoBioFactors vol 17no 1ndash4 pp 93ndash102 2003

[86] S Sai Kachout K J Hamza N Karray Bouraoui L C Leclercand Z Ouerghi ldquoSalt-induced changes in antioxidative enzymeactivities in shoot tissues of two Atriplex varietiesrdquo NotulaeBotanicae Horti Agrobotanici vol 41 pp 115ndash121 2013

[87] D P Dixon M Skipsey and R Edwards ldquoRoles for glutathionetransferases in plant secondary metabolismrdquo Phytochemistryvol 71 no 4 pp 338ndash350 2010

[88] P J Thornalley ldquoThe glyoxalase system new developmentstowards functional characterization of a metabolic pathwayfundamental to biological liferdquoBiochemical Journal vol 269 no1 pp 1ndash11 1990

[89] H El-Shabrawi B Kumar T Kaul M K Reddy S L Singla-Pareek and S K Sopory ldquoRedox homeostasis antioxidantdefense and methylglyoxal detoxification as markers for salttolerance in Pokkali ricerdquoProtoplasma vol 245 no 1 pp 85ndash962010

[90] K Nahar M Hasanuzzaman M M Alam and M FujitaldquoExogenous glutathione alleviates short-term abiotic stress bymodulating antioxidant defense and methylglyoxal detoxifica-tion system inV radiata seedlingsrdquo in Proceedings of the AnnualMain Meeting of the Society for Experimental Biology (SEB rsquo12)Salzburg Austria July 2012


[91] V S Reddy and S K Sopory ldquoGlyoxalase I fromBrassica junceamolecular cloning regulation and its over-expression confertolerance in transgenic tobacco under stressrdquoThe Plant Journalvol 17 no 4 pp 385ndash395 1999

[92] S L Singla-Pareek S K Yadav A Pareek M K Reddy andS K Sopory ldquoTransgenic tobacco overexpressing glyoxalasepathway enzymes grow and set viable seeds in zinc-spiked soilsrdquoPlant Physiology vol 140 no 2 pp 613ndash623 2006

[93] S L Singla-Pareek S K Yadav A Pareek M K Reddy andS K Sopory ldquoEnhancing salt tolerance in a crop plant byoverexpression of glyoxalase IIrdquo Transgenic Research vol 17 no2 pp 171ndash180 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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GenomicsInternational Journal of


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BioinformaticsAdvances in

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Evolutionary BiologyInternational Journal of



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Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


toxicity [7] At molecular level one of the common events inplants grown under salt stress is the considerable increases inreactive oxygen species (ROS) such as singlet oxygen (1O

2)

superoxide radical (O2

∙minus) hydrogen peroxide (H2O2) and

hydroxyl radical (∙OH) [2] However the production of ROSgreatly depends on the degree and duration of the impositionof stress and types of crop as well [2] Considering thedestructive effects of salt-induced oxidative stress in plants itis crucial to keep the ROS level below the toxic limit Plantsalways try to keep well-developed enzymatic and nonen-zymatic antioxidant defense system ready to encounter thedeleterious effects of ROS [2]The enzymatic system includesthe four enzymes of the ascorbate-glutathione (AsA-GSH)cycle ascorbate peroxidase (APX) monodehydroascorbatereductase (MDHAR) dehydroascorbate reductase (DHAR)and glutathione reductase (GR) as well as other enzymeslike superoxide dismutase (SOD) catalase (CAT) glutathioneperoxidase (GPX) and glutathione S-transferase (GST) Thenonenzymatic antioxidants include ascorbic acid (AsA) glu-tathione (GSH) phenolic compounds alkaloids nonproteinamino acids and 120572-tocopherols They act together in scav-enging or detoxifying ROS and subsequent protection ofplant cells from oxidative damage [2 4] However this systemacts differently in different plant species and cultivars andit was observed that the enhancement of the antioxidantdefense system is often correlated with salt stress tolerance [47] Methylglyoxal (MG) is another highly reactive cytotoxicwhich is produced largely under abiotic stress includingsalinity [14 15] and leads to damages to proteins lipids andDNA [16 17] In line with antioxidant defense system plantsalso possess glyoxalase system consisting of two enzymesglyoxalase I (Gly I) and glyoxalase II (Gly II) those candetoxify MG Enzymes of the glyoxalase system are found toregulate environmental stresses including salinity as reportedin many plant studies [2 3 10 18] It was reported that thecoordinated upregulation of both the antioxidant defense andglyoxalase systems is necessary to attain significant toleranceto oxidative stress [2 6]

It is an urgent task of plant biologists to explore suitablemechanisms of developing salt tolerant crop plants that canproduce sufficient yield under adverse condition In recentdecades many researchers have been trying to find the waysto alleviate salt stress or to overcome salt injury in plantsAmong them exogenous application of substances such asosmoprotectants phytohormones antioxidants and traceelements came to attention in recent times [4 19 20]

In response to various environmental stresses plantsdemonstrate a variety of adaptive mechanisms to counteractthem Since one of the primary responses under salt stressis osmotic adjustment compatible solutes such as proline(Pro) and glycine betaine (GB) are very common to beaccumulated during salt stress and play a fundamental rolein osmotic adjustment in plants [21] These compatiblesolutes are accumulated in the cytosol without disturbingintracellular biochemistry which ameliorate the detrimentaleffects of salinity [22ndash25]

In many plant species increased accumulation of Pro andGB were observed as an indicator of salt stress tolerance

[22 26] However most of the plants especially underelevated levels of salt cannot synthesize sufficient amountof these osmoregulators In many recent reports exogenousapplications of Pro and GB were found to act as protectantsunder salt stress [12 25 27] Besides osmoprotection Pro andGB also showed their roles in elimination of oxidative stressby triggering the antioxidant defense and also glyoxalasesystem [25 28ndash32]

Although there are several reports on the role of Pro andGB in salt stress tolerance in terms of growth and physiologyfew investigations have been done on the effects of exogenousPro and GB on both antioxidant defense and the glyoxalasesystem in rice Therefore we investigated the protectiveeffects of these osmoprotectants on the antioxidant defenseand glyoxalase systems in rice seedlings grown under salinemedia We also investigated the comparative performance oftwo modern rice varieties differing in their salt tolerance toknow their actual adaptive mechanisms under salt stress withor without osmoprotectants

2 Materials and Methods

21 Plant Materials and Stress Treatments Seeds of two rice(Oryza sativa L) cultivars cv BRRI dhan49 (salt sensitive)and cv BRRI dhan54 (salt tolerant) were collected fromBangladesh Rice Research Institute (BRRI) and surface-sterilized with 70 ethanol for 10min followed by washingseveral times with sterilized distilled water Seeds were thensoaked for 24 h in the dark Seeds were sown on plastic netsupon plastic beakers containing distilled water and kept inthe dark at 28 plusmn 2∘C for germination After 48 h uniformlygerminated seeds were transferred to growth chamber withcontrolled conditions (light intensity 100120583molmminus2 sminus1 tem-perature 25 plusmn 2∘C relative humidity 65ndash70) and duringthe growing period hyponex solution (Hyponex Japan) wasused as nutrient Two levels of salt stresses (150 and 300mMNaCl) were imposed on fourteen-day-old rice seedlings withwithout 5mM proline [l (-) Proline Wako Japan] andbetaine (Betaine Wako Japan) and where these protectantswere sprayed twice a daymixing with the wetting agent 002Tween 20 (Tween 20 Wako Japan) Control plants weregrownwithHyponex solution onlyDatawere taken after 48 hof NaCl treatment The experiment was repeated three times(119899 = 3) under the same conditions

22 Measurement of Relative Water Content Relative watercontent (RWC) was measured according to Barrs andWeath-erley [33] Leaf laminaswereweighed (freshwt FW) and thenimmediately floated on distilled water in a petri dish for 8 hin the dark Turgid weights (TW) were obtained after dryingexcess surface water with paper towels Dry weights (DW)were measured after drying at 80∘C for 48 h The calculationwas done using the following formula

RWC () = FW minus DWTW minus DW

times 100 (1)

23 Determination of Chlorophyll Content Chlorophyll con-tent was determined by homogenizing leaf samples (05 g)








4in













2O2-mediated pos-















2O2as


3 012mMNADPH






3 Results






a

cb b

dc

abA

BC A AB

DC

AB

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Relat

ive w

ater

cont

ent (

)



150and S


















a

cb b

d

b bA

BC ABC AB

DC C

000010020030040050060070080090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Chl a

(mgg

minus1

fresh

wei

ght)

(a)


a

cb b

d

b bA BC ABC AB

DC C

000

005

010

015

020

025

030

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Chl b

(mgg

minus1

fresh

wei

ght)

(b)

BC


a

cb b

d

b bA ABC AB

DC C

000

020

040

060

080

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Chl (a

+ b

) (m

ggminus1

fresh

wei

ght)

(c)


150and S









dd

abcbc c

aab

E

D

CB

C

AB

001020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Prol

ine (120583

mol

gminus1

fresh

wei

ght)


150and S









4 Discussion


2 O2






e

cd d

a

bcb

DC

D D

AB BC

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


MD

A (n

mol

gminus1

fresh

wei

ght)

(a)


e

b

cd d

a

cb

DC

D D

A

BC B

05

1015202530354045

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

LOX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


e

c

d d

a

bc b

C

B

C C

A

B B

02468

101214161820

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

H2O2

(nm

olgminus

1fre

sh w

eigh

t)

(c)



and S300






2O2is






2O2content was


a

b

a a

c

b

c

BCA A A

C

A AB

0

500

1000

1500

2000

2500

3000

3500

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


AsA

(nm

olgminus

1fre

sh w

eigh

t)

(a)

ac

ab bc

d d d

D

C

AB A

CB B

0

100

200

300

400

500

600

700

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

(nm

olgminus

1fre

sh w

eigh

t)

(b)

e

bc

d

c

a

b bc

D

C CC

A

B B

0102030405060708090

100

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSS

G (n

mol

gminus1

fresh

wei

ght)

(c)

a

c

b

c

d d d

B

D

C

A

E

D D

02468

10121416

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

GSS

G ra

tio

(d)


150and S







bc

de

ab

e

bcbc

C

A A AB

A A

0102030405060708090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


SOD

(uni

tmgminus

1pr

otei

n)

(a)

a

cb b

ed d

DBC

A AB

E

C C

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


CAT

(120583m

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S








2[4] The enhanced


2O2to





c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)


150and S






2O2scavenging role



2O2scavenging



a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








4in













2O2-mediated pos-















2O2as


3 012mMNADPH






3 Results






a

cb b

dc

abA

BC A AB

DC

AB

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Relat

ive w

ater

cont

ent (

)



150and S


















a

cb b

d

b bA

BC ABC AB

DC C

000010020030040050060070080090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Chl a

(mgg

minus1

fresh

wei

ght)

(a)


a

cb b

d

b bA BC ABC AB

DC C

000

005

010

015

020

025

030

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Chl b

(mgg

minus1

fresh

wei

ght)

(b)

BC


a

cb b

d

b bA ABC AB

DC C

000

020

040

060

080

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Chl (a

+ b

) (m

ggminus1

fresh

wei

ght)

(c)


150and S









dd

abcbc c

aab

E

D

CB

C

AB

001020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Prol

ine (120583

mol

gminus1

fresh

wei

ght)


150and S









4 Discussion


2 O2






e

cd d

a

bcb

DC

D D

AB BC

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


MD

A (n

mol

gminus1

fresh

wei

ght)

(a)


e

b

cd d

a

cb

DC

D D

A

BC B

05

1015202530354045

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

LOX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


e

c

d d

a

bc b

C

B

C C

A

B B

02468

101214161820

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

H2O2

(nm

olgminus

1fre

sh w

eigh

t)

(c)



and S300






2O2is






2O2content was


a

b

a a

c

b

c

BCA A A

C

A AB

0

500

1000

1500

2000

2500

3000

3500

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


AsA

(nm

olgminus

1fre

sh w

eigh

t)

(a)

ac

ab bc

d d d

D

C

AB A

CB B

0

100

200

300

400

500

600

700

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

(nm

olgminus

1fre

sh w

eigh

t)

(b)

e

bc

d

c

a

b bc

D

C CC

A

B B

0102030405060708090

100

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSS

G (n

mol

gminus1

fresh

wei

ght)

(c)

a

c

b

c

d d d

B

D

C

A

E

D D

02468

10121416

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

GSS

G ra

tio

(d)


150and S







bc

de

ab

e

bcbc

C

A A AB

A A

0102030405060708090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


SOD

(uni

tmgminus

1pr

otei

n)

(a)

a

cb b

ed d

DBC

A AB

E

C C

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


CAT

(120583m

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S








2[4] The enhanced


2O2to





c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)


150and S






2O2scavenging role



2O2scavenging



a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







2O2as


3 012mMNADPH






3 Results






a

cb b

dc

abA

BC A AB

DC

AB

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Relat

ive w

ater

cont

ent (

)



150and S


















a

cb b

d

b bA

BC ABC AB

DC C

000010020030040050060070080090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Chl a

(mgg

minus1

fresh

wei

ght)

(a)


a

cb b

d

b bA BC ABC AB

DC C

000

005

010

015

020

025

030

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Chl b

(mgg

minus1

fresh

wei

ght)

(b)

BC


a

cb b

d

b bA ABC AB

DC C

000

020

040

060

080

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Chl (a

+ b

) (m

ggminus1

fresh

wei

ght)

(c)


150and S









dd

abcbc c

aab

E

D

CB

C

AB

001020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Prol

ine (120583

mol

gminus1

fresh

wei

ght)


150and S









4 Discussion


2 O2






e

cd d

a

bcb

DC

D D

AB BC

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


MD

A (n

mol

gminus1

fresh

wei

ght)

(a)


e

b

cd d

a

cb

DC

D D

A

BC B

05

1015202530354045

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

LOX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


e

c

d d

a

bc b

C

B

C C

A

B B

02468

101214161820

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

H2O2

(nm

olgminus

1fre

sh w

eigh

t)

(c)



and S300






2O2is






2O2content was


a

b

a a

c

b

c

BCA A A

C

A AB

0

500

1000

1500

2000

2500

3000

3500

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


AsA

(nm

olgminus

1fre

sh w

eigh

t)

(a)

ac

ab bc

d d d

D

C

AB A

CB B

0

100

200

300

400

500

600

700

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

(nm

olgminus

1fre

sh w

eigh

t)

(b)

e

bc

d

c

a

b bc

D

C CC

A

B B

0102030405060708090

100

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSS

G (n

mol

gminus1

fresh

wei

ght)

(c)

a

c

b

c

d d d

B

D

C

A

E

D D

02468

10121416

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

GSS

G ra

tio

(d)


150and S







bc

de

ab

e

bcbc

C

A A AB

A A

0102030405060708090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


SOD

(uni

tmgminus

1pr

otei

n)

(a)

a

cb b

ed d

DBC

A AB

E

C C

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


CAT

(120583m

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S








2[4] The enhanced


2O2to





c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)


150and S






2O2scavenging role



2O2scavenging



a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


a

cb b

dc

abA

BC A AB

DC

AB

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Relat

ive w

ater

cont

ent (

)



150and S


















a

cb b

d

b bA

BC ABC AB

DC C

000010020030040050060070080090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Chl a

(mgg

minus1

fresh

wei

ght)

(a)


a

cb b

d

b bA BC ABC AB

DC C

000

005

010

015

020

025

030

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Chl b

(mgg

minus1

fresh

wei

ght)

(b)

BC


a

cb b

d

b bA ABC AB

DC C

000

020

040

060

080

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Chl (a

+ b

) (m

ggminus1

fresh

wei

ght)

(c)


150and S









dd

abcbc c

aab

E

D

CB

C

AB

001020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Prol

ine (120583

mol

gminus1

fresh

wei

ght)


150and S









4 Discussion


2 O2






e

cd d

a

bcb

DC

D D

AB BC

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


MD

A (n

mol

gminus1

fresh

wei

ght)

(a)


e

b

cd d

a

cb

DC

D D

A

BC B

05

1015202530354045

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

LOX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


e

c

d d

a

bc b

C

B

C C

A

B B

02468

101214161820

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

H2O2

(nm

olgminus

1fre

sh w

eigh

t)

(c)



and S300






2O2is






2O2content was


a

b

a a

c

b

c

BCA A A

C

A AB

0

500

1000

1500

2000

2500

3000

3500

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


AsA

(nm

olgminus

1fre

sh w

eigh

t)

(a)

ac

ab bc

d d d

D

C

AB A

CB B

0

100

200

300

400

500

600

700

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

(nm

olgminus

1fre

sh w

eigh

t)

(b)

e

bc

d

c

a

b bc

D

C CC

A

B B

0102030405060708090

100

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSS

G (n

mol

gminus1

fresh

wei

ght)

(c)

a

c

b

c

d d d

B

D

C

A

E

D D

02468

10121416

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

GSS

G ra

tio

(d)


150and S







bc

de

ab

e

bcbc

C

A A AB

A A

0102030405060708090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


SOD

(uni

tmgminus

1pr

otei

n)

(a)

a

cb b

ed d

DBC

A AB

E

C C

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


CAT

(120583m

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S








2[4] The enhanced


2O2to





c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)


150and S






2O2scavenging role



2O2scavenging



a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


a

cb b

d

b bA

BC ABC AB

DC C

000010020030040050060070080090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Chl a

(mgg

minus1

fresh

wei

ght)

(a)


a

cb b

d

b bA BC ABC AB

DC C

000

005

010

015

020

025

030

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Chl b

(mgg

minus1

fresh

wei

ght)

(b)

BC


a

cb b

d

b bA ABC AB

DC C

000

020

040

060

080

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Chl (a

+ b

) (m

ggminus1

fresh

wei

ght)

(c)


150and S









dd

abcbc c

aab

E

D

CB

C

AB

001020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Prol

ine (120583

mol

gminus1

fresh

wei

ght)


150and S









4 Discussion


2 O2






e

cd d

a

bcb

DC

D D

AB BC

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


MD

A (n

mol

gminus1

fresh

wei

ght)

(a)


e

b

cd d

a

cb

DC

D D

A

BC B

05

1015202530354045

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

LOX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


e

c

d d

a

bc b

C

B

C C

A

B B

02468

101214161820

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

H2O2

(nm

olgminus

1fre

sh w

eigh

t)

(c)



and S300






2O2is






2O2content was


a

b

a a

c

b

c

BCA A A

C

A AB

0

500

1000

1500

2000

2500

3000

3500

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


AsA

(nm

olgminus

1fre

sh w

eigh

t)

(a)

ac

ab bc

d d d

D

C

AB A

CB B

0

100

200

300

400

500

600

700

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

(nm

olgminus

1fre

sh w

eigh

t)

(b)

e

bc

d

c

a

b bc

D

C CC

A

B B

0102030405060708090

100

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSS

G (n

mol

gminus1

fresh

wei

ght)

(c)

a

c

b

c

d d d

B

D

C

A

E

D D

02468

10121416

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

GSS

G ra

tio

(d)


150and S







bc

de

ab

e

bcbc

C

A A AB

A A

0102030405060708090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


SOD

(uni

tmgminus

1pr

otei

n)

(a)

a

cb b

ed d

DBC

A AB

E

C C

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


CAT

(120583m

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S








2[4] The enhanced


2O2to





c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)


150and S






2O2scavenging role



2O2scavenging



a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


dd

abcbc c

aab

E

D

CB

C

AB

001020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Prol

ine (120583

mol

gminus1

fresh

wei

ght)


150and S









4 Discussion


2 O2






e

cd d

a

bcb

DC

D D

AB BC

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


MD

A (n

mol

gminus1

fresh

wei

ght)

(a)


e

b

cd d

a

cb

DC

D D

A

BC B

05

1015202530354045

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

LOX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


e

c

d d

a

bc b

C

B

C C

A

B B

02468

101214161820

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

H2O2

(nm

olgminus

1fre

sh w

eigh

t)

(c)



and S300






2O2is






2O2content was


a

b

a a

c

b

c

BCA A A

C

A AB

0

500

1000

1500

2000

2500

3000

3500

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


AsA

(nm

olgminus

1fre

sh w

eigh

t)

(a)

ac

ab bc

d d d

D

C

AB A

CB B

0

100

200

300

400

500

600

700

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

(nm

olgminus

1fre

sh w

eigh

t)

(b)

e

bc

d

c

a

b bc

D

C CC

A

B B

0102030405060708090

100

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSS

G (n

mol

gminus1

fresh

wei

ght)

(c)

a

c

b

c

d d d

B

D

C

A

E

D D

02468

10121416

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

GSS

G ra

tio

(d)


150and S







bc

de

ab

e

bcbc

C

A A AB

A A

0102030405060708090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


SOD

(uni

tmgminus

1pr

otei

n)

(a)

a

cb b

ed d

DBC

A AB

E

C C

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


CAT

(120583m

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S








2[4] The enhanced


2O2to





c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)


150and S






2O2scavenging role



2O2scavenging



a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


e

cd d

a

bcb

DC

D D

AB BC

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


MD

A (n

mol

gminus1

fresh

wei

ght)

(a)


e

b

cd d

a

cb

DC

D D

A

BC B

05

1015202530354045

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

LOX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


e

c

d d

a

bc b

C

B

C C

A

B B

02468

101214161820

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

H2O2

(nm

olgminus

1fre

sh w

eigh

t)

(c)



and S300






2O2is






2O2content was


a

b

a a

c

b

c

BCA A A

C

A AB

0

500

1000

1500

2000

2500

3000

3500

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


AsA

(nm

olgminus

1fre

sh w

eigh

t)

(a)

ac

ab bc

d d d

D

C

AB A

CB B

0

100

200

300

400

500

600

700

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

(nm

olgminus

1fre

sh w

eigh

t)

(b)

e

bc

d

c

a

b bc

D

C CC

A

B B

0102030405060708090

100

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSS

G (n

mol

gminus1

fresh

wei

ght)

(c)

a

c

b

c

d d d

B

D

C

A

E

D D

02468

10121416

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

GSS

G ra

tio

(d)


150and S







bc

de

ab

e

bcbc

C

A A AB

A A

0102030405060708090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


SOD

(uni

tmgminus

1pr

otei

n)

(a)

a

cb b

ed d

DBC

A AB

E

C C

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


CAT

(120583m

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S








2[4] The enhanced


2O2to





c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)


150and S






2O2scavenging role



2O2scavenging



a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


a

b

a a

c

b

c

BCA A A

C

A AB

0

500

1000

1500

2000

2500

3000

3500

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


AsA

(nm

olgminus

1fre

sh w

eigh

t)

(a)

ac

ab bc

d d d

D

C

AB A

CB B

0

100

200

300

400

500

600

700

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

(nm

olgminus

1fre

sh w

eigh

t)

(b)

e

bc

d

c

a

b bc

D

C CC

A

B B

0102030405060708090

100

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSS

G (n

mol

gminus1

fresh

wei

ght)

(c)

a

c

b

c

d d d

B

D

C

A

E

D D

02468

10121416

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GSH

GSS

G ra

tio

(d)


150and S







bc

de

ab

e

bcbc

C

A A AB

A A

0102030405060708090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


SOD

(uni

tmgminus

1pr

otei

n)

(a)

a

cb b

ed d

DBC

A AB

E

C C

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


CAT

(120583m

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S








2[4] The enhanced


2O2to





c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)


150and S






2O2scavenging role



2O2scavenging



a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


bc

de

ab

e

bcbc

C

A A AB

A A

0102030405060708090

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


SOD

(uni

tmgminus

1pr

otei

n)

(a)

a

cb b

ed d

DBC

A AB

E

C C

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


CAT

(120583m

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S








2[4] The enhanced


2O2to





c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)


150and S






2O2scavenging role



2O2scavenging



a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


c

b

a a

dc c

D

BCC

A

CB BC

000

020

040

060

080

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


APX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


bc

ab ab

c

a abCD

BA A

D

BB

01020304050607080

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

MD

HA

R (n

mol

minminus1

mgminus

1

prot

ein)

(b)


a

b

a a

c

b bB B

A A

C

AB

0

50

100

150

200

250

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

DH

AR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(c)


ab

aa

b

a a

C

B

AA

B

AA

0

20

40

60

80

100

120

140

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GR

(nm

olm

inminus1

mgminus

1pr

otei

n)

(d)


150and S






2O2scavenging role



2O2scavenging



a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


a

dabc

ab

e

cd bcB

A A A

CB B

0

20

40

60

80

100

120

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


GPX

(nm

olm

inminus1

mgminus

1pr

otei

n)

(a)


d

bc

aa

c

abab

D

AAB A

C BC

D

0

10

20

30

40

50

60

70

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

GST

(nm

olm

inminus1

mgminus

1pr

otei

n)

(b)


150and S



ac

a ab

dbc bc

D

C

AAB

C

B B

000005010015020025030035040045050

Con

trol

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B


Gly

I (120583

mol

minminus1

mgminus

1pr

otei

n)

(a)


dec

ab

e

ccdC

B

A AB B

B

000

005

010

015

020

025

030

035C

ontro

l

S150

S150

+ P

ro

S150

+ G

B

S300

S300

+ P

ro

S300

+ G

B

Gly

II (120583

mol

minminus1

mgminus

1pr

otei

n)

(b)


150and S






2O2











5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









5 Conclusion




Acknowledgments


References











































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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