Research Article Plant Growth-Promoting Rhizobacteria ...downloads.hindawi.com/journals/bmri/2016/6284547.pdf · ective bioresource for enhancing salt tolerance and growth of okra

Research ArticlePlant Growth-Promoting Rhizobacteria Enhance Salinity StressTolerance in Okra through ROS-Scavenging Enzymes

Sheikh Hasna Habib12 Hossain Kausar34 and Halimi Mohd Saud1

1Department of Agricultural Technology Faculty of Agriculture Universiti Putra Malaysia 43400 Serdang Selangor Malaysia2Oilseed Research Centre Bangladesh Agricultural Research Institute (BARI) Gazipur 1701 Bangladesh3Laboratory of Food Crops Institute of Tropical Agriculture Faculty of Agriculture Universiti Putra Malaysia43400 Serdang Selangor Malaysia4Department of Agroforestry and Environmental Science Sher-E-Bangla Agricultural University Dhaka Bangladesh

Correspondence should be addressed to Halimi Mohd Saud halimiupmedumy

Received 10 September 2015 Revised 29 December 2015 Accepted 30 December 2015

Academic Editor Qaisar Mahmood

Copyright copy 2016 Sheikh Hasna Habib 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

Salinity is a major environmental stress that limits crop production worldwide In this study we characterized plant growth-promoting rhizobacteria (PGPR) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and examined their effect onsalinity stress tolerance in okra through the induction of ROS-scavenging enzyme activity PGPR inoculated okra plants exhibitedhigher germination percentage growth parameters and chlorophyll content than control plants Increased antioxidant enzymeactivities (SOD APX and CAT) and upregulation of ROS pathway genes (CAT APX GR and DHAR) were observed in PGPRinoculated okra plants under salinity stress With some exceptions inoculation with Enterobacter sp UPMR18 had a significantinfluence on all tested parameters under salt stress as compared to other treatments Thus the ACC deaminase-containing PGPRisolate Enterobacter sp UPMR18 could be an effective bioresource for enhancing salt tolerance and growth of okra plants undersalinity stress

1 Introduction

Soil salinity is a major problem in agriculture that limitsplant growth and causes significant loss of crop productivityworldwide [1 2] Salinity affects up to 20 and 50 of thetotal cultivated and irrigated land in the world respectively[3]However the use of salinewater in agriculture is graduallyincreasing owing to shortage of fresh water Consequently onone hand salt-affected areas are constantly increasing andon the other hand a significant amount of arable land is beingabandoned every year because of salinity [4]

Okra (Abelmoschus esculentus L) is an annual vegetablecrop cultivated in tropical and subtropical regions It isconsidered a high-value vegetable crop owing to its highlevels of vitamins minerals carbohydrates and fats [5]Although it has good nutritional value as well as high con-sumer demand the yield of okra per hectare is very lowand this lower productivity arises mainly from soil salinity

Salt deposits in the crop field are a result of the use ofsaline underground irrigation water Discharge of industrialeffluents into irrigation canals is also a potential source of saltsin agricultural soil Saline water reduces the transpirationrate of plants by disrupting the evapotranspiration systemthus reducing crop yield [6] A high percentage of salt inthe root zone affects root density root turgor pressure andwater absorption which eventually affects plant growth anddevelopment The okra plant is sensitive to salinity especiallyin the early stage of its growth [7] where salinity affects waterand nutrient uptake of the plant and ionic stress reducesleaf expansion Altered morphological traits in the canolaplant [8] reduced plant dry matter and leaf area in soybeans[9] and reduced yield of canola [10] due to salinity havebeen reported In the root zone high salt concentrationdecreases soil water potential and water availability whichcauses dehydration at the cellular level eventually leading toosmotic stress [11]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 6284547 10 pageshttpdxdoiorg10115520166284547

2 BioMed Research International

Salinity stress generates reactive oxygen species (ROS)namely H

2O2 Ominus2 and OHminus that damage the DNA RNA

and proteins [12 13] These ROS compounds also causechlorophyll destruction and damage the root meristem activ-ity [14] Antioxidant enzymes such as superoxide dismutase(SOD) catalase (CAT) and ascorbate peroxidase (APX) havethe ability to scavenge the ROS and maintain them at lowlevels Superoxide dismutase is ametalloenzyme that plays animportant role in protecting cells from oxidative damage bycatalyzing the conversion of the superoxide radical to H

2O2

[15 16] Ascorbate peroxidase has vital defensive role againstROS [17] and can catalyze the breakdown of H

2O2that is

produced by SOD Catalase reduces ROS levels by catalyzingthe breakdown of H

2O2into H

2O and O

2[12 13]

Genetic engineering is an attractive approach that cangenerate plants resistant to salt stress [18] AtNHX1 overex-pressing transgenic Brassica napus plants were found to growflower and produce seeds under 200mM salt stress [19]However the transgenic approach is time-consuming andexpensive ACC deaminase-containing PGPR can reduce thedeleterious effects of environmental stress and can enhancestress tolerance of plants by a variety of mechanisms suchas the synthesis of phytohormones mineral solubilizationnutrient uptake increased leaf area increased chlorophylland soluble protein content and antioxidant enzyme activi-ties [20] Ethylene is important for plant growth and devel-opment as well as in the fruit ripening process but anexcess amount of ethylene might decrease seed germinationand root growth [21 22] It is also reported that ethyleneproduction increases under stress conditions and results ininhibitory effects on plants [23] However ACC deaminase-containing PGPR can hydrolyse ACC the precursor ofethylene thereby reducing the excess ethylene and rescueplants from inhibitory effects [24ndash26]

The plant growth-promoting effects of the interactions ofPseudomonas fluorescensYsS6 P migulae 8R6 and their ACCdeaminase deficient mutants on the growth of tomato plantswere investigated under 165mM and 185mM of salt stressThe plants treated with ACC deaminase-containing bacterialisolates exhibited higher fresh and dry biomass higherchlorophyll content and a greater number of flowers andbuds than the ACC deaminase deficient bacteria and controlplants [27] Similarly Saravanakumar and Samiyappan [22]reported that the ACC deaminase-containing P fluorescensstrain TDK1 increased the vigor index of groundnut seedlingssignificantly under 120mM of salt stress condition as com-pared to plants pretreated with a Pseudomonas strain lackingACCdeaminase activity ACCdeaminase-containingBacillussubtilis also induced salinity stress tolerance in hydropon-ically grown tomato plants [28] Bacteria containing highamounts of SOD and CAT play an important protective roleagainst the deleterious effect of ROS under stress conditions[16 29] Moreover induction of salt stress tolerance usingPGPR is an efficient and inexpensive method However thereare very few reports on PGPR induced salinity tolerancein the okra plant caused by changes in ROS-scavengingenzymes Therefore in this study we characterized the ACCdeaminase-containing PGPR with respect to its effect on

growth antioxidant enzyme activities and expression profilesof ROS pathway genes in the okra plant under high salt stress

2 Materials and Methods

21 Source of Plant Growth-Promoting Rhizobacteria FifteenPGPR isolates were used in this study All PGPR isolateswere initially isolated from crop fields of the UniversitiPutra Malaysia (UPM) Serdang Selangor and SemerakPasir Puteh Kelantan Malaysia by using the dilution platetechnique All selected PGPR isolates possessed nitrogenfixation phosphate solubilization indoleacetic acid (IAA)synthesis and salt tolerant properties [30]

22 ACC Deaminase Activity of Plant Growth-PromotingRhizobacteria All 15 PGPR isolates were tested for ACCdeaminase activity following the method described by Jacob-son et al [31] Bacterial isolates were grown onNutrient Broth(NB Difcotrade) for 48 h at 28 plusmn 2∘C The cultures were diluted10-fold with sterileMgSO

4(01M) solution ACC (3mM)was

filter sterilized with 02 120583m filter membrane and stored atminus20∘C until further use In a 96-well plate 120 120583L of minimalsalt medium (MSM) was added to each well To the first 4lanes 15 120583L of MgSO

4(01M) was added and to the next 4

lanes 15 120583L of (NH4)2SO4(01M) was added Thawed ACC

(15 120583L) was added to the remaining 4 lanes Each of the PGPRisolates (15 120583L) was added into each well separately For theuntreated control 15 120583L of MgSO

4(01M) was used instead

of the PGPR culture All plates were incubated for 48 h andthe optical density (OD) was measured at 600 nm with theThermo Scientific Multiskantrade GO microplate spectropho-tometer (Thermo Fisher Scientific Inc USA)

The OD values of ACC- and (NH4)2SO4-containing

wells were compared with the MgSO4-containing wells to

determine the ability of PGPR isolates to utilize ACC for theirgrowthThePGPR isolates were categorized into three groupsas isolates with higher medium and lower ACC utilizingrate depending upon the ratio of their OD values at 600 nmfor ACC substrate as compared to (NH

4)2SO4 Isolates with

higher ACC-utilization rate showed OD values for wells withACC substrate close to OD values for wells with (NH

4)2SO4

in the initial 48 h of growth Similarly isolates with mediumACC-utilization rate showed lower OD values for ACC wellsas compared to those for (NH

4)2SO4in the initial 48 h of

growth Isolateswith lowerACC-metabolic rate possessed thelowest OD values for ACC wells (close to OD values of wellswith MgSO

4) in the same time

23 Identification of PGPR Isolates PGPR isolates UPMR2and UPMR18 with the highest ACC-metabolizing activitieswere identified by 16S rRNA gene sequencing as Bacillusmegaterium and Enterobacter sp respectively (Figure 1(a))In brief each PGPR isolate was grown at 28∘C for 48ndash72 hin 5mL of NB Of each culture 1mL was transferred intoseparate 15mL microcentrifuge tubes Bacterial cells werepelleted by centrifugation at 12000 g for 5min (EppendorfCentrifuge 5810 R Hamburg Germany) Bacterial genomicDNA was extracted with the Genomic DNA Mini Kit

BioMed Research International 3

1 2 M

1400bp

(a)

Bacillus nealsonii KC329823Bacillus circulans strain KM349203

UPMR2Bacillus megaterium KF956591UPMR18Enterobacter sp GQ871449

Enterobacter sp LC014954Bacillus sp AB425363

100

100

10073

78

01

(b)

Figure 1 (a) Agarose gel electrophoresis of 16S rDNA PCR products of bacterial isolates LaneM 100 bP DNA ladder Lanes 1 and 2 bacterialisolates UPMR2 and UPMR18 (b) The neighbor-joining tree shows the phylogenetic relationship of the isolates UPMR2 and UPMR18 withrelated isolates from the NCBI database

(Yeastern Biotech Co Ltd) according to the manufacturerrsquosprotocol

The universal primer pairs 27f (51015840-AGAGTTTGATCM-TGGCTCAG-31015840) and 1492R (51015840-GGTTACCTTGTTACG-ACTT-31015840) were used to amplify the 16S rRNA coding regionThe 25 120583L PCR reaction mixture consisted of the followingcomponents 1 120583L of each extracted PGPR genomic DNA015 120583M of each primer 1x PCR reaction buffer (FermentasUSA) 02mM of dNTP mix 25U of Taq polymerase(Fermentas USA) 25mM of MgCl

2 and nucleic acid-

free water to make up final volume The thermal cyclingconditions were as follows one cycle for 5min at 94∘C30 cycles at 94∘C for 30 sec 55∘C for 30 sec and 72∘C for1min followed by an additional cycle at 72∘C for 5min Theamplified PCR products were purified using QIAquick GelExtraction Kit (QIAGEN Inc USA) and sent for sequencingThe obtained sequences were analyzed and compared withsequences obtained from the GenBank database using theBLAST program to determine the percent similarity Amolecular phylogenetic tree was constructed inMega version4 software following the neighbor-joining method [32] TheBLASTX analysis showed that the isolates UPMR2 andUPMR18 matched with B megaterium and Enterobacter sprespectively with 99 similarity Phylogenetic analysis ofthe bacterial isolates was also performed on the basis of theneighborhood joining method with 100 bootstrap samplingreplicates (Figure 1(b))

24 Evaluation of PGPR Isolates on Okra Seed Germinationunder Salt Stress Two PGPR isolates UPMR2 and UPMR18were selected based on their growth promotion halotoler-ance and ACC deaminase activities and were investigated inthe context of seed germination in okra under different levelsof NaCl stress

Seeds of the local Malaysian okra variety five anchorwere used in this study The seeds were surface sterilizedwith 05 (vv) sodium hypochlorite for 20min followed byrepetitive washes with distilled water The seeds were thensoaked in PGPR suspension (108 cfumL) or in distilled water(control) for 24 h A total of 10 seeds were sown in Petri dishes(9mmdiameter) with two sheets ofWhatman number 1 filterpapers The seeds were moistened either with distilled water(control) or with solutions of varying NaCl concentration

(25 50 75 and 100mM) and kept at 25∘C in the darkAfter three days the emergence of the radicle from the seedwas considered as an index of germination The germinationpercentage was calculated as follows

Germination = (No of seeds germinated

No of seeds sown) times 100 (1)

25 Evaluation of PGPR Isolates on Growth Promotion ofOkra under Salt Stress Okra seeds were surface sterilizedand sown in Petri dishes After emergence of the radicleand plumule the seedlings were planted in plastic pots (oneseedlingpot) filled with sterilized soil The soil belongs toSerdang soil series which was sandy-loam in texture (orderUltisols) The soil contained total N (078) available P(5492 ppm) potassium (218 ppm) and total C (294) andhad a pH of 532 Twenty-five milliliters of PGPR suspension(108 cfumL) was applied to the roots of potted okra plantsat 15 days after transplantation (DAT) and 22 DAT Controlplants were treated with the same amount of water Theplants were subjected to salt stress two weeks after the secondinoculation by watering with 75mM of NaCl solution Toprevent the plants from undergoing osmotic shock NaClconcentration was imposed in 25mM increments per dayuntil the final concentrationwas attained after three days [33]The soil surface was covered with black plastic to preventsalt loss Four treatment groups were defined as follows T0noninoculated salt treated plants T1 UPMR2 inoculated salttreated plants T2UPMR18 inoculated salt treated plants andT3 both UPMR2 and UPMR18 inoculated salt treated plantsEach treatment group comprised 16 plants Plants were grownfor 15 days under salt stress conditions Growth parametersthat is plant height root length freshweight of leaf stem androot and dry weight of leaf stem and root were recorded at15 days after salt treatment

251 Leaf Chlorophyll Measurements The total leaf chloro-phyll and chlorophyll a (chla) and chlorophyll b (chlb)content and the chlorophyll ab ratio were determined at15 days after salt treatment by using the method describedby Moran and Porath [34] Frozen leaflet samples of 02 gfrom each treatment were cut into small pieces and placedin a glass vial containing 2mL of NN-dimethylformamide


(DMF) and were covered with aluminum foil The vials werethen incubated at 4∘C for 48 h The absorbance readings ofchlorophyll a and b were taken at wave lengths of 663 nm and645 nm respectively with a UV-visible spectrophotometer(Genesys 10UV Thermo Fisher Scientific USA) using DMFas a blank The concentrations of total chlorophyll chla andchlb were calculated according to the following equations[35]

Chlorophyll a = (00127119863663minus 000269119863

645)

Chlorophyll b = (00229119863645minus 00468119863

663)

Total chlorophyll = Chlorophyll a + Chlorophyll b

(2)

where 119863663

is absorbance at 663 nm wave length and 119863645

isabsorbance at 645 nm wave length

252 Enzymatic Assay Leaf protein was extracted fromfrozen leaflet samples ground in liquid nitrogen using ice-cold mortar and pestle Protein was extracted in 3mL ofextraction buffer containing 100mM K-phosphate buffer(pH 78) 01mM EDTA 14mM 2-mercaptoethanol and01 (vv) Triton X-100 for SOD (EC 11511) activity or50mM K-phosphate buffer (pH 70) 2mM EDTA 20mMascorbate and 01 (vv) Triton X-100 for CAT (EC 11116)and APX (EC 111111) activities The homogenate was thancentrifuged at 15000timesg for 15min at 4∘C The supernatantwas used for total protein [36] and enzymatic assays

SOD activity was determined according to the methoddescribed by Giannopolitis and Ries [37] The reactionmixture contained 50mM K-phosphate buffer (pH 78)01mM EDTA 13mM methionine 75120583M nitrobluetetra-zolium (NBT) 2120583M riboflavin and 100 120583L enzyme extractSOD activity was determined by the ability of the enzyme toinhibit photochemical reduction of NBT on blue formazanfollowed by monitoring absorbance of the reaction mixtureat 560 nm

The total CAT activity in the leaf was assayed based on therate of H

2O2consumption at 240 nm [38] The assay mixture

of 3mL contained 100mMphosphate buffer (pH 70) 01mMEDTA 01 H

2O2 and 20120583L enzyme extract After addition

of the enzyme extract to the reaction mixture decrease inH2O2levels was determined by measuring the absorbance at

240 nm with a UV1000 spectrophotometer and quantified byusing the extinction coefficient (36M21 cm21)

Total leaf APX activity was estimated at 290 nm by themethod described by Chen and Asada [39] The 3mL APXassay mixture contained 50mM K-phosphate buffer (pH70) 01mM H

2O2 05mM ascorbate and 20 120583L enzyme

extract The amount of ascorbate oxidized was calculatedusing extinction coefficient 119864 = 28mMminus1 cmminus1

253 Expression of ROS Pathway Genes Total RNA wasextracted from approximately 01 g of frozen okra leaf sam-ples using TRIzol reagent (Invitrogen USA) The first-strand cDNA was synthesized from 1120583g of total RNAusing QuantiTect Reverse Transcription Kit (Qiagen USA)according to the manufacturerrsquos instructions The expression

Table 1 Primer pairs used for RT-PCR in this study

NCBIaccessionnumber

Primername Sequence (51015840-31015840)

X55749 Actin F CTGGTGGTGCAACAACCTTAR GAATGGAAGCAGCTGGAATC

AB041343 APX F ACCAATTGGCTGGTGTTGTTR TCACAAACACGTCCCTCAAA

AY442179 CAT F TGCCCTTCTATTGTGGTTCCR GATGAGCACACTTTGGAGGA

X76533 GR F GGATCCTCATACGGTGGATGR TTAGGCTTCGTTGGCAAATC

DQ512964 DHAR F AGGTGAACCCAGAAGGGAAAR TATTTTCGAGCCCACAGAGG

patterns of genes of the ROS pathway (APX CAT DHARand GR) were analyzed by semiquantitative RT-PCR usingthe primer pairs [40] mentioned in Table 1 with actin as acontrol

RT-PCR was performed using aliquots of 1120583L cDNAsamples The PCR conditions were set as follows one cycle at95∘C for 5min followed by 35 cycles at 95∘C for 30 sec 52∘Cfor 30 sec and 72∘C for 15 sec followed by an additional cycleof five minutes at 72∘CThe PCR products were separated on15 agarose gel and were observed under UV light

26 Statistical Analysis All experiments were conductedusing completely randomized design (CRD) with four repli-cates The data were subjected to analysis of variance(ANOVA) and tested for significance using the least signif-icant difference (LSD) by PC-SAS software (SAS InstituteCary NC USA 2001)

3 Results

31 ACC Deaminase Activity The ACC deaminase activityof the PGPR isolates was determined qualitatively to char-acterize the isolates for their ability to use ACC as the solenitrogen source The PGPR isolates were categorized intothree groups as isolates with higher (gt07) medium (05ndash069) and lower (lt05) ACC metabolism rate depending ontheir growth which was measured in terms of cell densityat OD

600(data not shown) On the basis of their high ACC

deaminase activity twoPGPR isolatesBmegateriumUPMR2andEnterobacter spUPMR18were selected to assess their salttolerance potency in okra plants

32 Effect of PGPR Isolates on Germination of Okra Seedsunder Salt Stress The results showed that the germinationof okra seeds was significantly (119901 le 005) affected by thePGPR isolates under different salt concentrations (Table 2)Both the PGPR isolates either separately or in combinationshowed 100 seed germination up to 75mM NaCl con-centrations Seed germination declined to 80 at 100mMNaCl concentration On the other hand in the noninoculatedgroup seed germination percentage was 70 at 75mMNaCl


Table 2 Effect of PGPR inoculation on germination of okra seedsunder salt stress

Treatments Salinity levels0mM 25mM 50mM 75mM 100mM

Control 100 100 100 70b 50b

Bacillusmegaterium(UPMR2) 100 100 100 100a 80a

Enterobacter sp(UPMR18) 100 100 100 100a 80a

Coinoculation ofUPMR2 amp UPMR18 100 100 100 100a 80a

Means within columns with the same letters are not significantly different at119901 lt 005

concentrationwhile the lowest germination percentage (50)was recorded at 100mM NaCl concentration

33 Effect of PGPR Isolates on Growth of Okra Seedlingsunder Salt Stress The effects of PGPR inoculation on okraplants were assessed at 75mM NaCl concentration Theresults demonstrate that the application of PGPR isolatesB megaterium UPMR2 and Enterobacter sp UPMR18 sig-nificantly (119901 le 005) increased shoot and root growth ofokra as compared to noninoculated plants Significantly (119901 le005) the highest leaf fresh (404 g) and dry (0469 g) weightwere obtained in Enterobacter sp UPMR18 (T2) treatedplants compared to other treatments As with leaf biomasssignificantly (119901 le 005) highest shoot dry weight (028 g) androot fresh weight (078 g) were also observed in the plantsreceiving Enterobacter sp UPMR18 (T2) under salt stress Inaddition significantly (119901 le 005) highest shoot fresh weightand root dry weight were recorded both in Enterobacter spUPMR18 (T2) treatment and in the combined application ofB megaterium UPMR2 and Enterobacter sp UPMR18 (T3)treated plants under salinity stress conditions (Table 3)

34 Determination of Okra Leaf Chlorophyll Content Thedata obtained for plant pigments showed that chlorophyll awas significantly (119901 le 005) highest in plants treated withEnterobacter sp UPMR18 (T2) and the combined applicationof both the PGPR (T3) compared to other treatmentsChlorophyll b and chlorophyll ab ratio were increasedsignificantly (119901 le 005) in all inoculated plants comparedto noninoculated salinized plants Similarly all the PGPRtreated plants showed significantly (119901 le 005) higher valuesfor total chlorophyll content than the control (Figure 2)

35 Determination of Activities of ROS-Scavenging EnzymesAll PGPR inoculated plants exhibited higher percent increasein ROS-scavenging enzymes in comparison to the noninocu-lated control plants (Figure 3) APX activity was significantly(119901 le 005) highest in B megaterium UPMR2 (T1) inoculatedsalinized plants where it was approximately 13 times higherthan the noninoculated plants Significantly (119901 le 005) higherCAT activity was also observed in all PGPR inoculated plantsas compared to control CAT activity was recorded to beapproximately 54 48 and 34 times higher in the plants

inoculated with Enterobacter sp UPMR18 (T2) combinedPGPR (T3) application and B megaterium UPMR2 (T1)respectively as compared to control plants under salt stressSimilarly the activity of SOD was also increased significantly(119901 le 005) (approximately 2 and 15 times higher) in theplants inoculated with Enterobacter sp UPMR18 (T2) andwith the combined application of both PGPR (T3) comparedto the noninoculated stressed plants

36 Expression Analysis of ROS Pathway Genes by RT-PCRThe ROS-scavenging enzymes CAT SOD and APX wereassessed in okra plants after salinity stress after inocu-lation or without inoculation with PGPR B megateriumUPMR2 and Enterobacter sp UPMR18The PGPR inoculatedsalinized plants exhibited the maximum percent increasein ROS-scavenging enzymes with respect to the noninocu-lated salinized control plants APX activity was significantlyincreased in B megaterium UPMR2 inoculated salinizedplants and was approximately 13 times higher than thatof noninoculated salinized plants (Figure 4) Significantlyincreased CAT activity was also observed in all PGPR inoc-ulated salinized plants than that of control (noninoculatedsalinized plants) CAT activity was recorded as approxi-mately 34 54 and 48 times higher in plants inoculatedwith B megaterium UPMR2 Enterobacter sp UPMR18and combined application of PGPR isolates respectivelycompared to that of noninoculated plants under the samestress conditions (Figure 4) Similarly SOD activity was alsoincreased significantly (approximately 2 and 15 times higher)in plants inoculated with Enterobacter sp UPMR18 and withcombined application of PGPR respectively growing understress condition compared to noninoculated salt stressedplants (Figure 4)

4 Discussion

Application of ACC deaminase-containing PGPR as a soilamendment resulted in enhanced seed germination chloro-phyll content and growth of okra plants under salinity stressby maintaining low stress ethylene levels and increasing theROS-scavenging enzymes Ethylene a plant growth regulatoris involved in various physiological responses [41] Howeverit is regarded as a stress hormone since it is synthesized ata rapid rate under stress [42] Stress ethylene decreases seedgermination and root development and eventually hindersplant growth [21 22] Microorganisms synthesizing the ACCdeaminase enzyme can cleave ACC to 120572-ketobutyrate andammonia thereby decreasing ethylene stress in plants [43ndash45] In this study two PGPR B megaterium UPMR2 andEnterobacter sp UPMR18 that possessed characteristics ofACC deaminase activity demonstrated their effectiveness ininducing salt tolerance and consequent improvement in thegrowth of okra plants under salt stress

Okra is a salt sensitive crop especially in its early growthstage [46] Seed germination of okra was reduced at a higherrate with increasing level of salinity in the noninoculatedgroup (Table 2) Salinity increases the osmotic potentialof growth medium and as a result seeds require moreenergy to absorb water resulting in decreased germination


Table 3 Effect of PGPR inoculation on growth attributes of okra plants under salinity stress

Treatment LFWplant LDWplant SFWplant SDWplant RFWplant RDWplant(g) (g) (g) (g) (g) (g)

T0 263 plusmn 013c 026 plusmn 003c 157 plusmn 014c 014 plusmn 002c 034 plusmn 003d 004 plusmn 001b

T1 283 plusmn 015c 028 plusmn 003c 190 plusmn 008b 016 plusmn 002c 047 plusmn 005c 003 plusmn 000b

T2 392 plusmn 017a 046 plusmn 001a 256 plusmn 029a 028 plusmn 001a 079 plusmn 008a 007 plusmn 002a

T3 328 plusmn 025b 037 plusmn 001b 265 plusmn 007a 022 plusmn 001b 065 plusmn 006b 006 plusmn 000a

T0 noninoculated salt treated plants T1 UPMR2 inoculated salt treated plants T2 UPMR18 inoculated salt treated plants and T3 UPMR2 and UPMR18inoculated salt treated plants LFW leaf fresh weight LDW leaf dry weight SFW shoot fresh weight SDW shoot dry weight RFW root fresh weight andRDW root dry weight Means within columns with the same letters are not significantly different at 119901 lt 005

TreatmentsT0 T1 T2 T3

c

b

aa

000

050

100

150

200

250

300

350

400

450

Chlo

roph

yll a

mg

g fre

sh le

af


b

a aa

000

050

100

150

200

250

300

Chlo

roph

yll b

mg

g fre

sh le

af


c

b

aa

000

020

040

060

080

100

120

140

160

180

200

Chlo

roph

yll a

b m

gg

fresh

leaf


c

ba a

0000

1000

2000

3000

4000

5000

6000

7000

Tota

l chl

orop

hyll

mg

g fre

sh le

af

Figure 2 Effect of PGPR inoculation on chlorophyll content of okra leaves under salt stress T0 noninoculated salt treated plants T1 UPMR2inoculated salt treated plants T2 UPMR18 inoculated salt treated plants and T3 both UPMR2 and UPMR18 inoculated salt treated plantsError bars refer to standard error of means of four replicates Means within columns with the same letters are not significantly different at119901 lt 005

[47 48] Our results were consistent with the results ofprevious studies [21 22] These studies demonstrated thatincreased salt concentrations decrease seed germination androot growth in dicotyledonous plants In addition ger-mination of Limonium stocksii and Suaeda fruticosa seedswas inhibited with increasing NaCl concentrations [49] Incontrast when the seeds were inoculated with the PGPRsuspension seed germination was reduced at a lower ratedespite increasing salinity This may indicate that the ACCdeaminase-containing PGPR isolatesBmegateriumUPMR2andEnterobacter spUPMR18 are able to ameliorate the effectof NaCl on growth medium The PGPR got attached to theseed surface and synthesized phytohormones in responseto amino acids produced by the seeds perhaps alleviating

the salinity stress [50] Our results were also supported by thefindings of Jalili et al [51] who demonstrated that the rateof germinating canola seeds (Brassica napus L) inoculatedwith ACC deaminase-containing plant growth-promotingP fluorescens and P putida was significantly higher undersalinity stress

ACCdeaminase-containing PGPR inoculated okra plantsshowed higher root and shoot biomass than noninoculatedplants under salinity stress This might be due to thepresence of PGPR isolates in the growth medium whichalleviate the effects of salinity on okra plants by producingACC deaminase ACC deaminase-containing plant growth-promoting bacteria have been documented to facilitate thegrowth of a variety of plants under high salinity conditions



c

d

b

aA

PX ac

tivity

0

100

200

300

400

500

600


c

d

ba

CAT

activ

ity

0

300

600

900

1200


cc

b

a

SOD

activ

ity

0

10

20

30

40

50

60

70

80

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)

Figure 3 Effect of PGPR inoculation on antioxidant enzymes activities in okra leaves under salt stress T0 noninoculated salt treated plantsT1 UPMR2 inoculated salt treated plants T2 UPMR18 inoculated salt treated plants and T3 both UPMR2 and UPMR18 inoculated salttreated plants Error bars refer to standard error of means of four replicates APX ascorbate peroxidase CAT catalase and SOD superoxidedismutase Means within columns with the same letters are not significantly different at 119901 lt 005

[52] Cucumber plants inoculated with wild-type P putidaUW4 and Gigaspora rosea BEG9 showed significantly higherroot and shoot fresh biomass than the plants that receivedACC deaminase deficient bacteria and untreated controlplants under 72mM salt stress [53] When canola seeds wereinoculated with ACC deaminase-containing salt tolerantbacteria under the 150mM NaCl condition the biomass oftreated plants increased by up to 47 of the control plants[54] Red pepper seedlings inoculated with ACC deaminase-containing salt tolerant bacteria reduced 57 stress ethyleneproduction and produced similar amounts of biomass as tothose in no salt treatment control plant [55]

Leaf chlorophyll concentration is an indicator of salttolerance and responds to increasing salinity [56] Chloro-phyll content was significantly higher in okra plants receivingbacterial suspension compared with control plant In controlplants chlorophyll is destroyed due to excessive amount ofsalts ions (Na and Cl) or reactive oxygen species (ROS)which disturb the cellular metabolism and result in thedegeneration of cell organelles in the leaf tissue [57 58]On the other hand the inoculated salt stressed okra plantsexhibited higher chlorophyll content and dark green leavesowing to the presence of ACC deaminase-containing PGPRisolates that maintain the photosynthetic efficiency of plantsby reducing ethylene biosynthesis Ali and colleagues [27]

observed that inoculation of plants with wild-type P flu-orescens YsS6 and P migulae 8R6 significantly increasedthe total chlorophyll content of tomato plants comparedwith their ACC deaminase deficient mutants and controlplants under salt stress Higher chlorophyll content was alsoreported in ACC deaminase-containing PGPR inoculatedsalt stressed rice [59] and cucumber [60] compared tononinoculated plants

Salinity stress leads to the formation of ROS namelysuperoxide (O

2

minus) singlet oxygen (O2) hydroxyl (OHminus) and

hydrogen peroxide (H2O2) which cause severe damage to

cell structures by exerting oxidation of cell membranes ina process known as oxidative stress [17 61] However adefensive system called the antioxidant enzyme system isalso activated under stress conditionsThis system consists ofseveral ROS-scavenging enzymes such as superoxide dismu-tase (SOD) peroxidase (POD) glutathione reductase (GR)monohydroascorbate reductase (MDHAR) ascorbate perox-idase (APX) and catalase (CAT) These antioxidant enzymeshave the ability to remove the free radicals produced duringabiotic stress conditions in the cell [13 58 62]The okra plantsinoculated with ACC deaminase-containing PGPR exhibitedsignificant elevation of antioxidant enzyme activities (APXCAT and SOD) compared to noninoculated plants undersaline conditions (Figure 3) thus confirming that PGPR


Actin

DHAR

GR

APX

CAT

T3 T2 T1 T0

Figure 4 Effect of PGPR inoculation onDHARGRAPX andCATtranscript levels by semiquantitative RT-PCR analysis of okra leavesunder salt stress T0 noninoculated salt treated plants T1 UPMR2inoculated salt treated plants T2 UPMR18 inoculated salt treatedplants and T3 both UPMR2 and UPMR18 inoculated salt treatedplants Actin positive control APX ascorbate peroxidase CATcatalase GR glutathione reductase and DHAR dehydroascorbatereductase

inoculated plants were adapted to saline conditions by elim-inating ROS through APX CAT and SOD activities Ourresults are also supported by the findings of Gururani etal [63] who reported enhanced activities of different ROS-scavenging enzymes in PGPR inoculated potato plants understress [63] Moreover in our study semiquantitative RT-PCRresults (Figure 4) revealed that expression levels of differentROS pathway genes encoding CAT APX GR and DHARwere increased in the ACC deaminase-containing PGPRtreated salinized plants compared to the untreated controlsThis result confirmed that the plants acquired protectionfrom salt challenge as a consequence of PGPR inoculationOur results were in agreement with the findings of Gururaniet al [63] who reported enhanced mRNA expression ofdifferent ROS pathway genes under salt drought and heavy-metal stress in PGPR inoculated potato plants

5 Conclusion

Little is known about enhanced salinity tolerance in okradue to ACC deaminase-containing PGPR The current studyshowed that ACC deaminase-containing Enterobacter spUPMR18 emerged as the best treatment agent for enhancingseed germination and growth of okra seedlings under salinitystress This may perhaps be due to a reduction in thegrowth inhibitory effect of salt on okra plants through theenhanced activity of antioxidant enzymes and expressionof ROS pathway genes induced by the PGPR ThereforeEnterobacter sp UPMR18 could be used for okra cultivation

in areas where salinity is amajor constraint However furtherresearch is required to validate the effectiveness of thisPGPR isolate Enterobacter sp UPMR18 in field conditionsbefore recommendation for large scale okra cultivation at theagricultural level

Conflict of Interests

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

Acknowledgment

The authors thank the Universiti Putra Malaysia for financialand technical support through the research Grant ldquoGeran(GP-IBT20139407000) Universiti Putra Malaysiardquo

References

[1] S I Allakhverdiev A Sakamoto Y Nishiyama M Inabaand N Murata ldquoIonic and osmotic effects of NaCl-inducedinactivation of photosystems I and II in Synechococcus sprdquoPlantPhysiology vol 123 no 3 pp 1047ndash1056 2000

[2] R Munns ldquoComparative physiology of salt and water stressrdquoPlant Cell amp Environment vol 25 no 2 pp 239ndash250 2002

[3] Z Cheng O Z Woody B J McConkey and B R GlickldquoCombined effects of the plant growth-promoting bacteriumPseudomonas putida UW4 and salinity stress on the Brassicanapus proteomerdquoApplied Soil Ecology vol 61 pp 255ndash263 2012

[4] W B FrommerU Ludewig andD Rentsch ldquoTaking transgenicplants with a pinch of saltrdquo Science vol 285 no 5431 pp 1222ndash1223 1999

[5] F N Emuh A E Ofuoku and E Oyefia ldquoEffect of intercrop-ping okra (Hibiscus esclentus)with pumpkin (CurcubitamaximaDutch ex Lam) on some growth parameters and economic yieldof maize (Zea mays) and maximization of land use in a fadamasoilrdquo Research Journal of Biological Sciences vol 1 no 1ndash4 pp50ndash54 2006

[6] LMDudley A Ben-Gal andU Shani ldquoInfluence of plant soiland water on the leaching fractionrdquo Vadose Zone Journal vol 7no 2 pp 420ndash425 2008

[7] A Cerda J Pardines M A Botella and V Martinez ldquoEffectof potassium on growth water relations and the inorganic andorganic solute contents for two maize cultivars grown undersaline conditionsrdquo Journal of Plant Nutrition vol 18 no 4 pp839ndash851 1995

[8] H M Zadeh and M B Naeini ldquoEffects of salinity stress on themorphology and yield of two cultivars of canola (Brassica napusL)rdquo Journal of Agronomy vol 6 no 3 pp 409ndash414 2007

[9] M R Amirjani ldquoEffect of salinity stress on growth mineralcomposition proline content antioxidant enzymes of soybeanrdquoAmerican Journal of Plant Physiology vol 5 no 6 pp 350ndash3602010

[10] A Bybordi and E Ebrahimian ldquoEffect of salinity stress onactivity of enzymes involved in nitrogen and phosphorousmetabolism case study canola (Brassica napus L)rdquo AsianJournal of Agricultural Research vol 5 no 3 pp 208ndash214 2011

[11] J Lloyd P E Kriedemann and D Aspinall ldquoComparativesensitivity of lsquoPrior Lisbonrsquo lemon and lsquoValenciarsquo orange treesto foliar sodium and chloride concentrationsrdquo Plant Cell ampEnvironment vol 12 no 5 pp 529ndash540 1989


[12] C A Jaleel K Riadh R Gopi et al ldquoAntioxidant defenseresponses physiological plasticity in higher plants under abioticconstraintsrdquoActa Physiologiae Plantarum vol 31 no 3 pp 427ndash436 2009

[13] R Mittler ldquoOxidative stress antioxidants and stress tolerancerdquoTrends in Plant Science vol 7 no 9 pp 405ndash410 2002

[14] J Foreman V Demidchik J H F Bothwell et al ldquoReactiveoxygen species produced by NADPH oxidase regulate plant cellgrowthrdquo Nature vol 422 no 6930 pp 442ndash446 2003

[15] J F Moran E K James M C Rubio G Sarath R V Klucasand M Becana ldquoFunctional characterization and expressionof a cytosolic iron-superoxide dismutase from cowpea rootnodulesrdquo Plant Physiology vol 133 no 2 pp 773ndash782 2003

[16] R Santos D Herouart A Puppo and D Touati ldquoCriticalprotective role of bacterial superoxide dismutase in Rhizobium-legume symbiosisrdquo Molecular Microbiology vol 38 no 4 pp750ndash759 2000

[17] K Apel and H Hirt ldquoReactive oxygen species metabolismoxidative stress and signal transductionrdquo Annual Review ofPlant Biology vol 55 pp 373ndash399 2004

[18] E Sergeeva S Shah and B R Glick ldquoGrowth of transgeniccanola (Brassica napus cv Westar) expressing a bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene onhigh concentrations of saltrdquoWorld Journal of Microbiology andBiotechnology vol 22 no 3 pp 277ndash282 2006

[19] H-X Zhang J N Hodson J P Williams and E BlumwaldldquoEngineering salt-tolerant Brassicaplants characterization ofyield and seed oil quality in transgenic plants with increasedvacuolar sodium accumulationrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 98 no22 pp 12832ndash12836 2001

[20] S Dobbelaere J Vanderleyden and Y Okon ldquoPlant growth-promoting effects of diazotrophs in the rhizosphererdquo CriticalReviews in Plant Sciences vol 22 no 2 pp 107ndash149 2003

[21] A A Belimov V I Safronova T A Sergeyeva et al ldquoChar-acterization of plant growth promoting rhizobacteria isolatedfrom polluted soils and containing 1-aminocyclopropane-1-carboxylate deaminaserdquo Canadian Journal of Microbiology vol47 no 7 pp 642ndash652 2001

[22] D Saravanakumar and R Samiyappan ldquoACC deaminase fromPseudomonas fluorescensmediated saline resistance in ground-nut (Arachis hypogea) plantsrdquo Journal of Applied Microbiologyvol 102 no 5 pp 1283ndash1292 2007

[23] J Yang J W Kloepper and C-M Ryu ldquoRhizosphere bacteriahelp plants tolerate abiotic stressrdquo Trends in Plant Science vol14 no 1 pp 1ndash4 2009

[24] S Mayak T Tirosh and B R Glick ldquoEffect of wild-type andmutant plant growth-promoting rhizobacteria on the rooting ofmung bean cuttingsrdquo Journal of Plant Growth Regulation vol 18no 2 pp 49ndash53 1999

[25] B R Glick C B Jacobson M K Schwarze and J J Pasternakldquo1-Aminocyclopropane-1-carboxylic acid deaminase mutantsof the plant growth promoting rhizobacterium Pseudomonasputida GR12-2 do not stimulate canola root elongationrdquo Cana-dian Journal of Microbiology vol 40 no 11 pp 911ndash915 1994

[26] B R Glick D M Penrose and J Li ldquoA model for the loweringof plant ethylene concentrations by plant growth-promotingbacteriardquo Journal ofTheoretical Biology vol 190 no 1 pp 63ndash681998

[27] S Ali T C Charles and B R Glick ldquoAmelioration of highsalinity stress damage by plant growth-promoting bacterial

endophytes that contain ACC deaminaserdquo Plant Physiology andBiochemistry vol 80 pp 160ndash167 2014

[28] M Woitke H Junge and W H Schnitzler ldquoBacillus subtilisas growth promotor in hydroponically grown tomatoes undersaline conditionsrdquo Acta Horticulturae vol 659 pp 363ndash3692004

[29] M Becana F J Paris L M Sandalio and L A Del RıoldquoIsoenzymes of superoxide dismutase in nodules of Phaseolusvulgaris L Pisum sativum L and Vigna unguiculata (L) walprdquoPlant Physiology vol 90 no 4 pp 1286ndash1292 1989

[30] S H Habib H M Saud M R Ismail and H KausarldquoBimolecular characterization of stress tolerant plant growthpromoting rhizobacteria (PGPR) for growth enhancement ofricerdquo International Journal of Agriculture and Biology In press

[31] C B Jacobson J J Pasternak and B R Glick ldquoPartialpurification and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the plant growth promoting rhi-zobacterium Pseudomonas putidaGR12-2rdquoCanadian Journal ofMicrobiology vol 40 no 12 pp 1019ndash1025 1994

[32] K Tamura J Dudley M Nei and S Kumar ldquoMEGA4 Molec-ular Evolutionary Genetics Analysis (MEGA) software version40rdquo Molecular Biology and Evolution vol 24 no 8 pp 1596ndash1599 2007

[33] M A Shahid M A Pervez R M Balal et al ldquoSalt stresseffects on somemorphological and physiological characteristicsof okra (Abelmoschus esculentus L)rdquo Soil and Environment vol30 no 1 pp 66ndash73 2011

[34] R Moran and D Porath ldquoChlorophyll determination in intacttissues using NN-dimethylformamiderdquo Plant Physiology vol65 no 3 pp 478ndash479 1980

[35] D I Arnon ldquoCopper enzymes in isolated chloroplasts Polyphe-nol oxidase in Beta vulgarisrdquo Plant Physiology vol 24 no 1 pp1ndash15 1949

[36] 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

[37] C N Giannopolitis and S K Ries ldquoSuperoxide dismutases IOccurrence in higher plantsrdquo Plant Physiology vol 59 no 2 pp309ndash314 1977

[38] E A Havir andN AMcHale ldquoBiochemical and developmentalcharacterization ofmultiple forms of catalase in tobacco leavesrdquoPlant Physiology vol 84 no 2 pp 450ndash455 1987

[39] G-X Chen and K Asada ldquoAscorbate peroxidase in tea leavesoccurrence of two isozymes and the differences in their enzy-matic and molecular propertiesrdquo Plant and Cell Physiology vol30 no 7 pp 987ndash998 1989

[40] Hemavathi C P Upadhyaya N Akula et al ldquoBiochemicalanalysis of enhanced tolerance in transgenic potato plantsoverexpressing d-galacturonic acid reductase gene in responseto various abiotic stressesrdquoMolecular Breeding vol 28 no 1 pp105ndash115 2011

[41] F B Abeles P W Morgan andM E J Salveit Ethylene in PlantBiology Academic Press San Diego Calif USA 2nd edition1992

[42] J C Stearns and B R Glick ldquoTransgenic plants with alteredethylene biosynthesis or perceptionrdquo Biotechnology Advancesvol 21 no 3 pp 193ndash210 2003

[43] B R Glick ldquoModulation of plant ethylene levels by the bacterialenzyme ACC deaminaserdquo FEMS Microbiology Letters vol 251no 1 pp 1ndash7 2005


[44] S Rashid T C Charles and B R Glick ldquoIsolation and char-acterization of new plant growth-promoting bacterial endo-phytesrdquo Applied Soil Ecology vol 61 pp 217ndash224 2012

[45] Y Sun Z Cheng and B R Glick ldquoThe presence of a 1-aminocyclopropane-1-carboxylate (ACC) deaminase deletionmutation alters the physiology of the endophytic plant growth-promoting bacterium Burkholderia phytofirmans PsJNrdquo FEMSMicrobiology Letters vol 296 no 1 pp 131ndash136 2009

[46] J Kamaluldeen I A M Yunusa A Zerihun J J Bruhl and PKristiansen ldquoUptake and distribution of ions reveal contrastingtolerance mechanisms for soil and water salinity in okra(Abelmoschus esculentus) and tomato (Solanum esculentum)rdquoAgricultural Water Management vol 146 pp 95ndash104 2014

[47] M Jamil and E H Rha ldquoThe effect of salinity (NaCI) on thegermination and seedling of sugar beet (Beta vulgaris L) andcabbage (Brassica oleracea L)rdquo Plant Resources vol 7 no 3 pp226ndash232 2004

[48] M Kafi and M Goldani ldquoEffect of water potential and type ofosmoticum on seed germination of three crop species of wheatsugarbeet and chickpeardquo Agricultural Sciences and Technologyvol 15 no 1 pp 121ndash133 2001

[49] A Hameed A Rasheed B Gul and M A Khan ldquoSalinityinhibits seed germination of perennial halophytes Limoniumstocksii and Suaeda fruticosa by reducing water uptake andascorbate dependent antioxidant systemrdquo Environmental andExperimental Botany vol 107 pp 32ndash38 2014

[50] C L Patten and B R Glick ldquoRole of Pseudomonas putidaindoleacetic acid in development of the host plant root systemrdquoApplied and Environmental Microbiology vol 68 no 8 pp3795ndash3801 2002

[51] F Jalili K Khavazi E Pazira et al ldquoIsolation and characteriza-tion of ACC deaminase-producing fluorescent pseudomonadsto alleviate salinity stress on canola (Brassica napus L) growthrdquoJournal of Plant Physiology vol 166 no 6 pp 667ndash674 2009

[52] Z A Zahir M Arshad and W T Frankenberger Jr ldquoPlantgrowth promoting rhizobacteria applications and perspectivesin agriculturerdquo Advances in Agronomy vol 81 pp 97ndash168 2001

[53] E Gamalero G Berta N Massa B R Glick and G LingualdquoInteractions between Pseudomonas putidaUW4 andGigasporarosea BEG9 and their consequences for the growth of cucumberunder salt-stress conditionsrdquo Journal of Applied Microbiologyvol 108 no 1 pp 236ndash235 2010

[54] M A Siddikee P S Chauhan R Anandham G-H Han andT Sa ldquoIsolation characterization and use for plant growthpromotion under salt stress of ACC deaminase-producinghalotolerant bacteria derived from coastal soilrdquo Journal ofMicrobiology and Biotechnology vol 20 no 11 pp 1577ndash15842010

[55] M A Siddikee B R Glick P S Chauhan W J Yimand T Sa ldquoEnhancement of growth and salt tolerance ofred pepper seedlings (Capsicum annuum L) by regulatingstress ethylene synthesis with halotolerant bacteria contain-ing 1-aminocyclopropane-1-carboxylic acid deaminase activityrdquoPlant Physiology and Biochemistry vol 49 no 4 pp 427ndash4342011

[56] G C Percival G A Fraser and G Oxenham ldquoFoliar salttolerance of Acer genotypes using chlorophyll fluorescencerdquoJournal of Arboriculture vol 29 no 2 pp 61ndash65 2003

[57] A B Hassine and S Lutts ldquoDifferential responses of saltbushAtriplex halimus L exposed to salinity and water stress inrelation to senescing hormones abscisic acid and ethylenerdquoJournal of Plant Physiology vol 167 no 17 pp 1448ndash1456 2010

[58] T Abbas M A Pervez C M Ayyub and R Ahmad ldquoAssess-ment of morphological antioxidant biochemical and ionicresponses of salttolerant and salt-sensitive okra (Abelmoschusesculentus) under saline regimerdquo Pakistan Journal of Life andSocial Sciences vol 11 no 2 pp 147ndash153 2013

[59] H B Bal L Nayak S Das and T K Adhya ldquoIsolation ofACC deaminase producing PGPR from rice rhizosphere andevaluating their plant growth promoting activity under saltstressrdquo Plant and Soil vol 366 no 1-2 pp 93ndash105 2013

[60] S-M Kang A L Khan M Waqas et al ldquoPlant growth-promoting rhizobacteria reduce adverse effects of salinity andosmotic stress by regulating phytohormones and antioxidantsin Cucumis sativusrdquo Journal of Plant Interactions vol 9 no 1pp 673ndash682 2014

[61] S Hussain A Khaliq A Matloob M A Wahid and I AfzalldquoGermination and growth response of three wheat cultivars toNaCl salinityrdquo Soil amp Environment vol 32 no 1 pp 36ndash43 2013

[62] GM Abogadallah ldquoDifferential regulation of photorespiratorygene expression by moderate and severe salt and drought stressin relation to oxidative stressrdquo Plant Science vol 180 no 3 pp540ndash547 2011

[63] M A Gururani C P Upadhyaya V Baskar J Venkatesh ANookaraju and S W Park ldquoPlant growth-promoting rhizobac-teria enhance abiotic stress tolerance in Solanum tuberosumthrough inducing changes in the expression of ROS-scavengingenzymes and improved photosynthetic performancerdquo Journal ofPlant Growth Regulation vol 32 no 2 pp 245ndash258 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Salinity stress generates reactive oxygen species (ROS)namely H

2O2 Ominus2 and OHminus that damage the DNA RNA

and proteins [12 13] These ROS compounds also causechlorophyll destruction and damage the root meristem activ-ity [14] Antioxidant enzymes such as superoxide dismutase(SOD) catalase (CAT) and ascorbate peroxidase (APX) havethe ability to scavenge the ROS and maintain them at lowlevels Superoxide dismutase is ametalloenzyme that plays animportant role in protecting cells from oxidative damage bycatalyzing the conversion of the superoxide radical to H

2O2

[15 16] Ascorbate peroxidase has vital defensive role againstROS [17] and can catalyze the breakdown of H

2O2that is

produced by SOD Catalase reduces ROS levels by catalyzingthe breakdown of H

2O2into H

2O and O

2[12 13]

Genetic engineering is an attractive approach that cangenerate plants resistant to salt stress [18] AtNHX1 overex-pressing transgenic Brassica napus plants were found to growflower and produce seeds under 200mM salt stress [19]However the transgenic approach is time-consuming andexpensive ACC deaminase-containing PGPR can reduce thedeleterious effects of environmental stress and can enhancestress tolerance of plants by a variety of mechanisms suchas the synthesis of phytohormones mineral solubilizationnutrient uptake increased leaf area increased chlorophylland soluble protein content and antioxidant enzyme activi-ties [20] Ethylene is important for plant growth and devel-opment as well as in the fruit ripening process but anexcess amount of ethylene might decrease seed germinationand root growth [21 22] It is also reported that ethyleneproduction increases under stress conditions and results ininhibitory effects on plants [23] However ACC deaminase-containing PGPR can hydrolyse ACC the precursor ofethylene thereby reducing the excess ethylene and rescueplants from inhibitory effects [24ndash26]

The plant growth-promoting effects of the interactions ofPseudomonas fluorescensYsS6 P migulae 8R6 and their ACCdeaminase deficient mutants on the growth of tomato plantswere investigated under 165mM and 185mM of salt stressThe plants treated with ACC deaminase-containing bacterialisolates exhibited higher fresh and dry biomass higherchlorophyll content and a greater number of flowers andbuds than the ACC deaminase deficient bacteria and controlplants [27] Similarly Saravanakumar and Samiyappan [22]reported that the ACC deaminase-containing P fluorescensstrain TDK1 increased the vigor index of groundnut seedlingssignificantly under 120mM of salt stress condition as com-pared to plants pretreated with a Pseudomonas strain lackingACCdeaminase activity ACCdeaminase-containingBacillussubtilis also induced salinity stress tolerance in hydropon-ically grown tomato plants [28] Bacteria containing highamounts of SOD and CAT play an important protective roleagainst the deleterious effect of ROS under stress conditions[16 29] Moreover induction of salt stress tolerance usingPGPR is an efficient and inexpensive method However thereare very few reports on PGPR induced salinity tolerancein the okra plant caused by changes in ROS-scavengingenzymes Therefore in this study we characterized the ACCdeaminase-containing PGPR with respect to its effect on

growth antioxidant enzyme activities and expression profilesof ROS pathway genes in the okra plant under high salt stress

2 Materials and Methods

21 Source of Plant Growth-Promoting Rhizobacteria FifteenPGPR isolates were used in this study All PGPR isolateswere initially isolated from crop fields of the UniversitiPutra Malaysia (UPM) Serdang Selangor and SemerakPasir Puteh Kelantan Malaysia by using the dilution platetechnique All selected PGPR isolates possessed nitrogenfixation phosphate solubilization indoleacetic acid (IAA)synthesis and salt tolerant properties [30]

22 ACC Deaminase Activity of Plant Growth-PromotingRhizobacteria All 15 PGPR isolates were tested for ACCdeaminase activity following the method described by Jacob-son et al [31] Bacterial isolates were grown onNutrient Broth(NB Difcotrade) for 48 h at 28 plusmn 2∘C The cultures were diluted10-fold with sterileMgSO

4(01M) solution ACC (3mM)was

filter sterilized with 02 120583m filter membrane and stored atminus20∘C until further use In a 96-well plate 120 120583L of minimalsalt medium (MSM) was added to each well To the first 4lanes 15 120583L of MgSO

4(01M) was added and to the next 4

lanes 15 120583L of (NH4)2SO4(01M) was added Thawed ACC

(15 120583L) was added to the remaining 4 lanes Each of the PGPRisolates (15 120583L) was added into each well separately For theuntreated control 15 120583L of MgSO

4(01M) was used instead

of the PGPR culture All plates were incubated for 48 h andthe optical density (OD) was measured at 600 nm with theThermo Scientific Multiskantrade GO microplate spectropho-tometer (Thermo Fisher Scientific Inc USA)

The OD values of ACC- and (NH4)2SO4-containing

wells were compared with the MgSO4-containing wells to

determine the ability of PGPR isolates to utilize ACC for theirgrowthThePGPR isolates were categorized into three groupsas isolates with higher medium and lower ACC utilizingrate depending upon the ratio of their OD values at 600 nmfor ACC substrate as compared to (NH

4)2SO4 Isolates with

higher ACC-utilization rate showed OD values for wells withACC substrate close to OD values for wells with (NH

4)2SO4

in the initial 48 h of growth Similarly isolates with mediumACC-utilization rate showed lower OD values for ACC wellsas compared to those for (NH

4)2SO4in the initial 48 h of

growth Isolateswith lowerACC-metabolic rate possessed thelowest OD values for ACC wells (close to OD values of wellswith MgSO

4) in the same time

23 Identification of PGPR Isolates PGPR isolates UPMR2and UPMR18 with the highest ACC-metabolizing activitieswere identified by 16S rRNA gene sequencing as Bacillusmegaterium and Enterobacter sp respectively (Figure 1(a))In brief each PGPR isolate was grown at 28∘C for 48ndash72 hin 5mL of NB Of each culture 1mL was transferred intoseparate 15mL microcentrifuge tubes Bacterial cells werepelleted by centrifugation at 12000 g for 5min (EppendorfCentrifuge 5810 R Hamburg Germany) Bacterial genomicDNA was extracted with the Genomic DNA Mini Kit


1 2 M

1400bp

(a)




100

100

10073

78

01

(b)




2 and nucleic acid-












645)


663)


(2)

where 119863663
















NCBIaccessionnumber










3 Results








Control 100 100 100 70b 50b











4 Discussion












c

b

aa

000

050

100

150

200

250

300

350

400

450

Chlo

roph

yll a

mg

g fre

sh le

af


b

a aa

000

050

100

150

200

250

300

Chlo

roph

yll b

mg

g fre

sh le

af


c

b

aa

000

020

040

060

080

100

120

140

160

180

200

Chlo

roph

yll a

b m

gg

fresh

leaf


c

ba a

0000

1000

2000

3000

4000

5000

6000

7000

Tota

l chl

orop

hyll

mg

g fre

sh le

af







c

d

b

aA

PX ac

tivity

0

100

200

300

400

500

600


c

d

ba

CAT

activ

ity

0

300

600

900

1200


cc

b

a

SOD

activ

ity

0

10

20

30

40

50

60

70

80

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)






2





Actin

DHAR

GR

APX

CAT

T3 T2 T1 T0



5 Conclusion





Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 M

1400bp

(a)




100

100

10073

78

01

(b)




2 and nucleic acid-












645)


663)


(2)

where 119863663
















NCBIaccessionnumber










3 Results








Control 100 100 100 70b 50b











4 Discussion












c

b

aa

000

050

100

150

200

250

300

350

400

450

Chlo

roph

yll a

mg

g fre

sh le

af


b

a aa

000

050

100

150

200

250

300

Chlo

roph

yll b

mg

g fre

sh le

af


c

b

aa

000

020

040

060

080

100

120

140

160

180

200

Chlo

roph

yll a

b m

gg

fresh

leaf


c

ba a

0000

1000

2000

3000

4000

5000

6000

7000

Tota

l chl

orop

hyll

mg

g fre

sh le

af







c

d

b

aA

PX ac

tivity

0

100

200

300

400

500

600


c

d

ba

CAT

activ

ity

0

300

600

900

1200


cc

b

a

SOD

activ

ity

0

10

20

30

40

50

60

70

80

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)






2





Actin

DHAR

GR

APX

CAT

T3 T2 T1 T0



5 Conclusion





Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




645)


663)


(2)

where 119863663
















NCBIaccessionnumber










3 Results








Control 100 100 100 70b 50b











4 Discussion












c

b

aa

000

050

100

150

200

250

300

350

400

450

Chlo

roph

yll a

mg

g fre

sh le

af


b

a aa

000

050

100

150

200

250

300

Chlo

roph

yll b

mg

g fre

sh le

af


c

b

aa

000

020

040

060

080

100

120

140

160

180

200

Chlo

roph

yll a

b m

gg

fresh

leaf


c

ba a

0000

1000

2000

3000

4000

5000

6000

7000

Tota

l chl

orop

hyll

mg

g fre

sh le

af







c

d

b

aA

PX ac

tivity

0

100

200

300

400

500

600


c

d

ba

CAT

activ

ity

0

300

600

900

1200


cc

b

a

SOD

activ

ity

0

10

20

30

40

50

60

70

80

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)






2





Actin

DHAR

GR

APX

CAT

T3 T2 T1 T0



5 Conclusion





Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Control 100 100 100 70b 50b











4 Discussion












c

b

aa

000

050

100

150

200

250

300

350

400

450

Chlo

roph

yll a

mg

g fre

sh le

af


b

a aa

000

050

100

150

200

250

300

Chlo

roph

yll b

mg

g fre

sh le

af


c

b

aa

000

020

040

060

080

100

120

140

160

180

200

Chlo

roph

yll a

b m

gg

fresh

leaf


c

ba a

0000

1000

2000

3000

4000

5000

6000

7000

Tota

l chl

orop

hyll

mg

g fre

sh le

af







c

d

b

aA

PX ac

tivity

0

100

200

300

400

500

600


c

d

ba

CAT

activ

ity

0

300

600

900

1200


cc

b

a

SOD

activ

ity

0

10

20

30

40

50

60

70

80

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)






2





Actin

DHAR

GR

APX

CAT

T3 T2 T1 T0



5 Conclusion





Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










c

b

aa

000

050

100

150

200

250

300

350

400

450

Chlo

roph

yll a

mg

g fre

sh le

af


b

a aa

000

050

100

150

200

250

300

Chlo

roph

yll b

mg

g fre

sh le

af


c

b

aa

000

020

040

060

080

100

120

140

160

180

200

Chlo

roph

yll a

b m

gg

fresh

leaf


c

ba a

0000

1000

2000

3000

4000

5000

6000

7000

Tota

l chl

orop

hyll

mg

g fre

sh le

af







c

d

b

aA

PX ac

tivity

0

100

200

300

400

500

600


c

d

ba

CAT

activ

ity

0

300

600

900

1200


cc

b

a

SOD

activ

ity

0

10

20

30

40

50

60

70

80

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)






2





Actin

DHAR

GR

APX

CAT

T3 T2 T1 T0



5 Conclusion





Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



c

d

b

aA

PX ac

tivity

0

100

200

300

400

500

600


c

d

ba

CAT

activ

ity

0

300

600

900

1200


cc

b

a

SOD

activ

ity

0

10

20

30

40

50

60

70

80

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)

(120583m

ol m

inminus1

mg

prot

einminus

1)






2





Actin

DHAR

GR

APX

CAT

T3 T2 T1 T0



5 Conclusion





Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Actin

DHAR

GR

APX

CAT

T3 T2 T1 T0



5 Conclusion





Acknowledgment


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





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Plant Growth-Promoting Rhizobacteria ...downloads.hindawi.com/journals/bmri/2016/6284547.pdf · ective bioresource for enhancing salt tolerance and growth of okra