BIOREMEDIATION OF DROUGHT STRESSED WHEAT (TRITICUM AESTIVUM L.) SEEDLINGS

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1 Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile BIOREMEDIATION OF DROUGHT STRESSED WHEAT (TRITICUM AESTIVUM L.) SEEDLINGS *Hnn M. Aou-Zeid 1 nd Slw A. Adel Ltif 1 Botny nd Miroiology Deprtment, Fulty of Siene, Alexndri University, Alexndri, Egypt Biology nd Geology Deprtment, Fulty of Edution, Alexndri University, Egypt *Author for Correspondene ABSTRACT A study ws onduted to evlute the effets of miroin iofertilizer, mixture of nitrogen-fixing nd phosphte-dissolving teri (Azototer sp. nd Azospirillium sp. nd Billus megterium) for meliorting negtive effets of drought stress on growth nd growth indies, photosyntheti effiieny, ntioxidnt enzymes, nd phytohormones of whet (Tritium estivum L.). Two levels of irrigtion (9% nd 5% of field pity) were employed in presene nd sene of iofertilizer. Whet seedling exhiited retrdtion of growth, redution of the photosyntheti effiieny, reltive wter ontent, nd memrne stility index, s well s the inrese of the ntioxidnt enzymes like superoxide dismutse, peroxidse, sorte peroxidse nd tlse, the prodution of mlondildehyde, hydrogen peroxide, nd the extensive impirments in ellulr ultr-struture in response to drought stress. Phytohormones indole-3 eti id (IAA), gierelli id (GA) ontents were deresed s result of wter defiit; onversely, sii id (ABA) showed mrked inrese. The intertion of miroin iofertilizer nd wter stress showed remrkle improving in growth hrteristis nd in some physiologil prmeters. The overll results suggest tht pplition of miroin iofertilizer inoultion llevites to some extent the deleterious effets of drought stress on whet seedlings. Keywords: Antioxidnt Enzymes, Biofertilizer, Photosyntheti Effiieny, Phytohormones, Wter Stress INTRODUCTION Drought is worldwide prolem, onstrining glol rop prodution seriously nd hs detrimentl effets on most of plnt growth stges suh s germintion, seedling growth, physiologil tivities nd ultimtely the plnt yield (Azimi et l., 13). Whet (Tritium estivum L.) is onsidered the most strtegi rop for Egypt nd mny other developing ountries. Whet should e irrigted when 5 55% of the ville soil wter is depleted in the root zone (Doorenos nd Pruitt, 199). Limittion of irrigtion wter resoures is widespred growing prolem for ultivtion of griulturl rops. When plnts re exposed to environmentl stresses suh s drought, retive oxygen speies (ROS) like, superoxide (O ), hydrogen peroxide (H O ), hydroxyl rdils (OH ) nd singlet oxygen (1O) re produed. The lne etween the prodution of ROS nd the quenhing tivity of the ntioxidnts is upset nd this often results in oxidtive dmge (Jung, 4). To redue the toxiity of ROS, plnt ells hve developed n nti oxidtive system, onsisting of protetive enzymes. Superoxide rdils re svenged y superoxide dismutse, while the resulting H O is redued to H O y tlse, peroxidse nd sorte peroxidse (Apel nd Hirt, 4). Plnts respond nd dpt to drought stress through vrious iohemil hnges inluding hnges of the endogenous hormone levels (Monneveux nd Belhssen, 1996). Plnts n reruit sisi id s n endogenous, signl to initite dptive responses (Wng et l., 8). However, the vrition of IAA nd GA ontents under drought stress re ontrditory. It ws reported tht drought resulted in derese of IAA ontent in the leves of whet (Xie et l., 3). However, Pustovoitov et l., (4) reported tht dpttion to drought ws ompnied with n inrese in the IAA ontent. Current soil mngement strtegies re minly dependent on inorgni hemil-sed fertilizers, whih used serious thret to humn helth nd environment. The exploittion of enefiil miroes s iofertilizer hs eome prmount importne in griulture setor for their potentil role in food sfety nd sustinle rop prodution (Bhrdwj et l., 14). Biofertilizers ontining miroorgnism, whih hve n ility to onvert nutritionlly importnt elements to ville form through iologil proesses Centre for Info Bio Tehnology (CIBTeh) 83

2 Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile (Vessey, 3). Wter nd nutrients re the most importnt ftors during plnt growth nd development. Defiit irrigtion nd use of iologil fertilizers re the ritil omponents to rop prodution in sustinle frming systems (Cnolt et l., 6). Miroorgnisms ould ply signifint role in stress mngement, lso provide exellent models for understnding stress tolerne mehnisms tht n e susequently engineered into rop plnts. The min ojetive of this study ws to investigte the effet of miroin iofertilizer on whet (Tritium estivum L.) growth, photosyntheti pigments, photohemil tivity of photo system II (PSII), ultr-struture, ntioxidnt defense system, in ddition to monitor the reltive wter ontent, lipid peroxidtion, nd memrne stility index, s well s the hnges in the endogenous level of phytohormones under norml irrigtion nd drought stress. MATERIALS AND METHODS Whet (Tritium estivum L.) v. Skh 93 ws used in this study, purhsed from the Interntionl Reserh Center, El-Dokki, Giz, Egypt. Grins were sujeted to surfe steriliztion with.1% sodium hypohlorite solution for 5min nd then rinsed severl times with distilled wter. Grin inoultion ws performed y mixing whet grins with the miroin using Ari gum s dhesive mteril. Miroin, ommeril multi-strins iofertilizer is produed y iofertilizers unit, Generl Orgniztion of Agriulture Equlizton Fund, Agriulture Reserh Centre, Ministry of Agriulture, Giz, Egypt. It is onstituted of mixture of N -fixing nd P-dissolving teri, the treted iofertilizer ontining N-free living teri (Azototer sp. nd Azospirillium sp.) nd phosphte dissolving teri (Billus megterium). The oted grins were then ir dried in shde for 3 minutes nd inoulted nd uninoulted grins were sown immeditely in pots ontining soil nd snd mixture (1:). The pots (in triplites) were pled in growth hmer under 14h light/1h drk yle t 3/18± C for dys. Pots were divided into two sets (inoulted nd uninoulted) oth irrigted with hlf strength Hoglnd s nutrient solution (Hoglnd nd Arnon, 195) till wter field pity. Drought stress tretment ws strted fter 5 dys from sowing, two tretments were imposed viz, 9%, nd 5% of the field pity, these tretments refleting onditions hieved s optimum level of wter supply (ontrol plnts) nd drought stress, respetively. Plnt Growth Prmeters At hrvest ( dys) plnt smples were olleted, in order to void dmging the roots when they pull out, ll of the pots were temporrily wterlogged for 1h. After refully uprooting, the plnt smples were divided into shoots nd roots. The smples were wshed severl times with distilled wter, nd the plnt shoot height nd root length (m) were mesured. To determine the dry weight, the shoots nd roots were oven-dried seprtely t 7 C for 7h. Fresh leves were used for different iohemil nlysis. Photosyntheti Pigment Contents The pigment frtions (hlorophyll, hlorophyll nd rotenoids) were estimted y the method desried y Lihtenthler (1987). Lef tissues (5mg) were homogenized in 1ml hilled etone (8%). The homogente ws entrifuged t 4,g for 1min. Asorne of the superntnt ws reorded t 663, 647 nd 47nm (using T8 UV Vis spetrophotometer - doule em). Chlorophyll Fluoresene Mesurements of hlorophyll fluoresene (Fv/Fm) were performed y OS-3P pulse modulted hlorophyll fluorimeter (Opti-sienes, Hudson, USA). Before every mesurement, leves were drkdpted for 3 min with lef-lips (Brnquinho et l., 1997). Estimtion of Photosyntheti Ativity (PS II) Chloroplst thylkoid memrnes (PS II memrnes) were isolted from whet leves ording to Arnon (1949) method. Photo system II tivity, s indited y the rte of,6-dihlorophenol indophenol (DCPIP) photo redution, ws monitored t 6nm using spetrophotometer s desried y Treest (197). Ultr-Struturl Anlysis Fresh tissues (1 mm ) tken from lst fully expnded leves were prepred ording to method of Loreto et l., (1) for eletron mirosopy proessing. The ultr-struture visuliztion nd Centre for Info Bio Tehnology (CIBTeh) 84

3 Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile photogrphing were rried out using the Trnsmission Eletron Mirosope (JEOL-TEM 1 CX) t the Eletron Mirosopi Unit, Fulty of siene, Alexndri University. Reltive Wter Content The reltive wter ontent ws estimted ording to Turner (1981( nd ws evluted from the eqution: RWC (%) = (FW DW) / (TW DW) x1, where FW is the fresh weight of the leves, TW is the weight t full turgor, mesured fter floting the leves for 4 hours in wter t room temperture nd DW is the weight estimted fter drying the leves for 4 hours t 8 º C or until onstnt weight is hieved. Memrne Stility Index Memrne stility index (MSI) ws determined y reording the eletril ondutivity of lef lehtes in doule distilled wter t 4 nd 1 º C. Lef smples (.1g) were ut into diss of uniform size nd tken in test tues ontining 1 ml of doule distilled wter in two sets. One set ws kept t 4 º C for 3 minutes nd nother set t 1 º C in oiling wter th for 15 minutes nd their respetive eletri ondutivities C 1 nd C were mesured y ondutivity meter (Deshmukh et l., 1991). Memrne stility index = [1- (C 1 /C )] x 1 Determintion of Mlondildehyde nd Hydrogen Peroxide Content The level of lipid peroxidtion produts ws estimted nd expressed s mlondildehyde (MDA) ording to Buege nd Aust (1978). The MDA onentrtions were expressed s μmol g -1 DW. Hydrogen peroxide levels were determined ording to Sergiev et l., (1997) nd the onentrtions were expressed s mmol g -1 DW. Extrtion nd Determintion of Antioxidnt Enzymes Ativities Fresh whet leves (1g) were homogenized in ie old 5mM phosphte uffer (ph 7.5) ontining.5mm EDTA with prehilled pestle nd mortr. The homogente ws entrifuged t 1,g for 1 min t 4 C nd the superntnts were olleted nd used for the determintion of different enzyme tivities. The protein ontent in the enzyme extrt ws determined ording to Hrtree (197). Superoxide dismutse (SOD) tivity ws determined following the method used y Ginnopolitis nd Ries (1977) where its ility to inhiit the photo redution of nitrolue tetrzolium (NBT) ws mesured. Ctlse (CAT) tivity ws ssyed with the method of Zhng et l., (5). One unit of enzyme determined the mount neessry to deompose 1μmol of H O per min t 5 C. Peroxidse (POD) tivity ws determined ording to Noreen nd Ashrf (9). Chnges in sorne of the retion solution t 47nm were determined fter every s. Asorte peroxidse (APX) tivity ws ssyed y the following redution in sorne t 9nm s sorte ws oxidized ording to the method of Boominthn nd Dorn (). The mixture ontined 5mM phosphte uffer (ph 7),.mM EDTA,.5mM sori id nd 5µg ovine serum lumin. Determintion of Plnt Phytohormons Hormones were extrted nd purified following Kettner nd Doerffling (1995) proedure. The plnt leves (1g) were ground in 8% methnol t 4 C for 7 hr with n ntioxidnt, utylted hydroxyl toluene. The extrted smple ws entrifuged nd the superntnt ws redued to queous phse using rotry evportor. The ph of queous phse ws djusted to.5-3. nd extrted four times with hlf volume of ethyl ette. The ethyl ette ws dried ompletely using rotry evportor nd the dried smple ws redissolved in 1ml of methnol (1%) nd ws nlyzed on HPLC (Agilent tehnologies 1 series) Exlipse XDB C18 (5 µm, 4.6 X 15 mm) olumn nd using UV detetor. Smples were injeted into HPLC olumn for identifition nd quntifition. Isortilly eluted t ml min -1 with methnol, % eti id nd H O (4::) s moile phses, detetion ws performed with n sorne monitor operting t 54 nm. Identifition nd pek ssignment of the ompound ws sed on omprison of its retention time with orresponding stndrd nd y spiking of smple with the stndrd. Centre for Info Bio Tehnology (CIBTeh) 85

4 RWC Length (m) Dry weight (g 5ind t-1 ) Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile Sttistil Anlysis Sttistil nlysis of the results ws rried out ording to Dunn s multiple rnge tests using SPSS-. Dt were sujeted to one-wy ANOVA following the method of Sokl nd Rohlf (1995). Differenes etween tretment-mens were onsidered sttistilly signifint t p.5. RESULTS AND DISCUSSION Results Dt represented in Figure 1 showed tht the whet plnts (Tritium estivum L.) inoulted with miroin iofertilizer, reorded signifint (P.5) inrese in ll plnt growth prmeters (shoot nd root lengths nd dry weights) nd photosyntheti pigments ontent ompring with uninoulted plnts in the norml irrigtion (ontrol plnts) or drought tretment. In generl, plnts were negtively ffeted y wter defiit tretment s ompred with norml irrigtion tretment. Wter stress ould restrit dry weight nd tissue wter ontent, lso shoot nd root lengths. The redution in shoot nd root lengths nd dry weights ws 35%, 4% nd 39%, 6% respetively, ompred to the ontrol. There ws redution in RWC, the mximum redution in RWC rehed out 5% ompred to the ontrol. Miroin inoulted plnts showed signifintly inrese in dry mtter, longer shoot nd root length in omprison with the drought stressed plnts (Figure 1B). 3 Shoot Root A Shoot Root B C D C+F D+F d Figure 1: Effet of Inoultion y Bteril Biofertilizer on Shoot nd Root, (A) Lengths, (B) Dry Weights nd (C) Lef RWC t Norml nd Drought Tretment of Whet Plnts. Vlues re Mens ±SD Bsed on Triplite Independent Determintions, nd Different Letters Mens Signifint Differene s Evluted y Dunn s Multiple Comprison Test; C: Norml Irrigtion (Control), D: Drought, C+F: Control+ Biofertilizer, D+F: Drought + Biofertilizer Chlorophylls nd rotenoids ontent were lso ffeted y drought stress; ontrol plnts exhiited higher totl hlorophyll ontent, the perent inhiition rehed 53% in drought stressed plnts. Applition of iofertilizer inreses to some extent the totl hlorophyll ontent, the inrese vlue rehed nerly out 1.-fold ompred to the ontrol. Crotenoids were insignifintly ffeted y pplition of iofertilizer, onversely, there ws signifint inrese in response to drought, nd the inrese vlue ws out 1.3- fold ompred to the ontrol (Figure A). In ddition, the rtio of Cr/Chl signifintly inresed under drought with respet to the ontrol nd the iofertilizer treted plnts (Figure B). The study of PSII Centre for Info Bio Tehnology (CIBTeh) 86 Tretment C

5 Fv/Fm PS II(µm DCPIP red. mgchl. -1 h- 1 ) mg g- 1 FW Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile photohemistry showed tht there ws mrked derese in the PSII nd in mximl quntum yield Fv/Fm of wter-stressed plnts ompred to the ontrol, the redution perentge ws out 4% nd 31% respetively. Applition of iofertilizer llevited the dverse effet of drought stress in whet leves (Figure C nd D). Compred with the ontrol, wter defiit tretment led to onsiderle ultr struturl ltertions in orgnelles. Fousing on the hloroplst, ontrol whet leves exhiited typil hloroplst struture, ellipsoidl shpe, with well displyed thylkoid memrnes orgnized in grnl nd stroml memrnes (Figure 3). In the orresponding tissues of the plnts sumitted to drought stress, the hloroplsts suffered severe dmge. Two types of hnges ould e distinguished, the first one ws the hloroplsts eme more rounded insted of n ellipsoidl shpe; nd the seond, there ws disruption of the memrne struture nd the integrity of the outer memrne nd the internl network of thylkoid memrnes ws lmost ompletely lost. During drought nd susequent iofertilizer reovery in the hloroplsts, slight hnges of the stroml nd grn lmelle region ould e oserved Chl. Chl. Chl.+ Chl. Crot. A d C D C+F D+F 14% 1% 18% 47% Cr/+ B C D C+F D+ F Tretment C Figure : Effet of Inoultion y Bteril Biofertilizer on Photosyntheti Pigments (A), Rtio etween Crotenoids nd Chlorophylls nd Photosynthesis Ativity {Fv/Fm (C) nd PSII (D)} t Norml Irrigtion nd Drought Tretment of Whet Plnts. Vlues re Mens ±SD Bsed on Triplite Independent Determintions nd Different Letters Mens Signifint Differene s Evluted y Dunn s Multiple Comprison Test; C: Norml Irrigtion (Control), D: Drought, C+F: Control+ Biofertilizer, D+F: Drought+ Biofertilizer Dt in Figure 4(A nd B) reveled tht the onentrtion of MDA, nd H O were signifintly inresed in the whet leves due to drought stress, vlues rehed out.5- nd 1.7- fold higher thn the ontrol. Tretment of iofertilizer ompnied with wter defiit signifintly deresed the MDA nd H O ontents ompred to the ontrol. Dt of MSI were represented in Figure 4C, ompred to the ontrol plnts; the MSI ws mrkedly deresed under drought ondition, while plnts previously inoulted with iofertilizer seemed to e meliorted. Simple liner regression ws otined in the present study (Figure 5A nd B) showed tht the oeffiient of determintion (R ) etween H O, MDA ontent in treted nd ontrol leves nd MSI were out.98 nd.881, respetively. Centre for Info Bio Tehnology (CIBTeh) Tretment D

6 H O (mmol g -1 DW) MDA (μmol g -1 DW) Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile Figure 3: Trnsmission Eletron Mirogrphs of Lef Mesophyll Cells from Norml Irrigtion nd Drought Tretment of Whet Plnts Inoulted y Bteril Biofertilizer nd Uninoulted; C: Norml Irrigtion (Control), D: Drought, D+F: Drought + Biofertilizer, (Th) Thylkoid, (CM) Chloroplst Memrne, (M) Mitohondri, (CW) Cell Wll A B Tretment Tretment MSI C 4% 9% 9% Figure 4: Effet of Inoultion y Bteril Biofertilizer on (A) H O ), (B) MDA Content nd (C) MSI t Norml Irrigtion nd Drought Tretment of Whet Plnts. Vlues re Mens ±SD Bsed on Triplite Independent Determintions, nd Different Letters Mens Signifint Differene s Evluted y Dunn s Multiple Comprison Test; C: Norml Irrigtion (Control), D: Drought, C+F: Control+ Biofertilizer, D+F: Drought + Biofertilizer Centre for Info Bio Tehnology (CIBTeh) 88 18% C D C+F D+ F

7 H O (mmol g -1 DW) MDA (μmol g -1 DW) Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile y = -.13x +.98 R² = A y = -.58x R² = B MSI MSI Figure 5: Regression Anlysis etween the HO, (A) MDA (B) nd MSI Results in Figure 6 illustrted tht drought stress used signifint inrese of SOD, POD CAT, nd APX speifi tivities, the vlues were -, 1.7-, 1.6-, nd 1.6-fold higher thn tht of the ontrol. Dt of plnts under well wtered onditions, inoulted with iofertilizer showed tivities nerly similr to tht of the ontrol. On the other hnd plnts inoulted with iofertilizer nd stressed showed redution in the speifi tivities of ll tested ntioxidnt enzymes ompred to the drought stressed ones. Exept for sisi id, the IAA nd GA ontents of whet leves were inhiited y rtio of 8% nd 46% respetively due to wter defiit ompred to the ontrol. Applition of iofertilizer in well wtered plnts enhned oth IAA nd GA, the mount ws inresed out 1.8- nd 1.-fold, respetively ompred with uninoulted plnts. Asii id ws mrkedly inresed under drought stress; the mount ws 5-fold greter thn ontrol. In leves treted with iofertilizer nd grown under drought ondition the ABA ontent ws rehed 35% less ompred with drought stressed leves (Figure 7). Units mg -1 protein min SOD μmol H O mg 1 protein min CAT OD 47 mg -1 protein min POD As 9 mg -1 protein min APX Tretment Tretment Figure 6: Effet of Inoultion y Bteril Biofertilizer on Antioxidnt Enzymes Ativities (SOD, CAT, POD, nd APX) t Norml Irrigtion nd Drought Tretment of Whet Plnts. Vlues re Mens ±SD Bsed on Triplite Independent Determintions, nd Different Letters Mens Signifint Differene s Evluted y Dunn s Multiple Comprison Test; C: Norml Irrigtion (Control), D: Drought, C+F: Control + Biofertilizer, D+F: Drought + Biofertilizer Centre for Info Bio Tehnology (CIBTeh) 89

8 µg g -1 FW Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile 3.5 IAA GA ABA Tretment Figure 7: Effet of Inoultion y Bteril Biofertilizer on Phytohormons (IAA, GA, nd ABA) t Norml Irrigtion nd Drought Tretment of Whet Plnts. Vlues re Mens ±SD Bsed on Triplite Independent Determintions, nd Different Letters Mens Signifint Differene s Evluted y Dunn s Multiple Comprison Test; C: Norml Irrigtion (Control), D: Drought, C+F: Control + Biofertilizer, D+F: Drought + Biofertilizer Disussion Different teril strins re eing used in ioremedition proesses, in this study, miroin mixed iofertilizer ws used to determine its effet to improve whet plnt tolerne for drought stress. Among studied tretments the miroil inoultions ws signifint nd the highest promoting effet oserved in well irrigted tretment ompred with the uninoulted stressed ones. Aording to the results, there ws mrked derese in growth prmeters, shoot nd root lengths, nd dry weight, RWC nd the totl hlorophyll ontent under drought stress ondition ompred with norml wter supply tretment (ontrol plnts). In onsensus, Khlil nd Yousef (14) reported tht, the redution in plnt growth in response to wter stress my e due to loking up of xylem nd phloem vessels thus, hindering ny trnslotion through them. Also, drought uses losses in tissue wter ontent whih redue turgor pressure in ell, therey inhiiting enlrgement nd division of ell using of redue of plnt growth nd dry mss umultion (Delfine et l., ). In this investigtion, the mximum photohemil effiieny of PSII nd the rtio Fv/Fm were estimted, their mounts were deresed under drought stress. The ellulr ultr-struturl nlysis illustrted tht the hloroplsts eme rounded, with disintegrting envelope nd disrupted nd irregulrly shped grn lmelle (Figure 3), this indites serious dmge to hloroplst struture, onsistent with Btr et l., (14) who reported tht drought stress uses not only sustntil dmge to photosyntheti pigments, ut it lso leds to deteriortion of thylkoid memrnes. Under previling study, the rotenoids (Cr) ontent nd Cr/Chl rtio were inresed in response to drought stress, this my referred to protetive role of rotenoids in photosynthesis; photo protetion nd their ntioxidnt tivity. Drought used onsiderle oxidtive stress y umultion of ROS (Mittler, ). In present study the tivities of SOD, POD, CAT, nd APX enzymes in whet leves were inresed s result of wter stress. These results re in good ord with the study of Rohmn et l., (16) who found tht enzymes like SOD, POD nd APX were involved in svenging ROS in mize in reds under drought stress. Dt showed tht the MDA ontent ws signifintly higher in stressed leves this my e referred to the indution of memrne lipid per oxidtion y mens of ROS our results were in greement with Mouss nd Aziz (8). The lower memrne stility index reflets the extent of lipid peroxidtion, whih in turn is onsequene of higher oxidtive stress due to wter stress onditions, our dt on H O ontent lso support these findings (Figure 4). In ordne with our results Buhnn et l., () onfirmed tht wter stress used wter loss from plnt tissues whih seriously impir oth memrne struture nd funtion. The results of this study lso indited signifint derese of IAA nd GA prodution nd inrese in ABA in the leves due to drought stress tht might e responsile, t lest prtly, for redution of the root nd shoot dry weight. Sleem et l., (7) delred tht, the over prodution of ABA in response to ioti nd ioti stresses leds to Centre for Info Bio Tehnology (CIBTeh) 9

9 Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile inhiition of root growth nd, onsequently, inhiition of the plnt growth under stress onditions. Hu et l., (1) reported tht ABA my indue ntioxidnt defense systems nd suppress toxiity of ROS under drought stress. Results showed tht the effet of intertion etween wter stress nd iofertilizer (miroin) tretment ws evluted positively. The present results re in greement with those otined y Heidri nd Golpyegni (1) who found tht inoultion with rhizoteri ould e effiiently used to improve growth, ntioxidnt sttus nd photosyntheti pigments under wter stress. They lso reported tht signifint inrese ws found in the inoultion with growth promoting rhizoteri, espeilly omintion of the teril speies. Bsed on the results, the lower vlues of MDA nd H O in response to pplition of miroin omined with wter defiit indite tht t ellulr level whet plnts re equipped with n effiient free rdil quenhing system tht offers protetion ginst oxidtive stress. Moreover, miroil inoultion ffet positively towrd IAA nd GA umultion nd redution in ABA ontent ompred to the stressed leves. The improve of the photosyntheti pity of whet treted with miroin iofertilizer might e due to the inrese of the vilility of phosphorus, nitrogen nd other nutrients, this ppered to oinide with the positive effets on the dry mss umultion. In greement with our results Grí-Frile et l., (15) reported tht, teril iofertilizers n improve plnt growth through the synthesis of plnt nutrients or phytohormones, whih n e sored y plnts. Likewise, Selvkumr et l., (1) reported tht, iofertilizer signifintly improved hlorophyll onentrtion, this is euse, nitrogen is the hief onstituent of protein, essentil for the formtion of protoplsm, whih leds to ell enlrgement, ell division nd ultimtely resulting in inresed plnt growth. Furthermore, Aou-Zeid (14) reported tht, the photosyntheti pity of mize treted with iofertilizer inresed due to the inrese of intrellulr mronutrients suh s K, C, Mg, nd P tht might enhne hlorophyll iosynthesis nd protet the photosyntheti systems. Miroin ontins Azototer sp. nd Azospirillium sp. nd Billus megterium. Cohen et l., (15) delred tht s ompenstion to root exudtes sereted y plnts, the miroorgnism plnt ssoition my improve plnt growth y synthesis of vitmins, ntiiotis, enzymes nd phytohormones, Azospirillum sp not only generte plnt growth promoting hormones, ut lso sprinkle hormones suh s ABA in stress onditions. (Mrulnd-Aguirre et l., (8), oserved n inresed level of hlorophyll ontent in Ltu stiv inoulted with Billus megterium, this ould e inditing the positive intertion of B. megterium this intertion might trigger the hlorophyll relted enzymes for hlorophyll synthesis nd leds to inrese the photosynthesis; lso, plnt-teril intertion lters the rohydrte metolisms of plnts. Reently, Esmeili et l., (16) stted tht the dequte supplies of the mironutrients nd iofertilizer ontining Azototer will hve fvorle effets on soyen qulity nd growth. Conlusion From the otined results, it n e onluded tht the dul pplition of phosphorus nd nitrogen iofertilizer resulted in improve nd overome the depressive effet of wter stress on whet plnt. Moreover, iofertilizers re importnt omponent in integrted nutrients mngements, they re ost effetive, eo-friendly nd renewle soure of plnt nutrients. From the present work the dt showed tht the miroin iofertilizer improved some physiologil prmeters of the whet plnt. Biofertilizer ould ply n importnt role in dpttion strtegies nd inrese of tolerne to ioti stresses suh s drought in griulturl plnts. They might reple prt of the use of hemil fertilizers, redues mount nd ost of hemil nd thus prevents the environment pollution from extensive pplition of hemil fertilizers. REFERENCES Aou-Zeid HM (14). The promotive role of lgl iofertilizer on the growth of mize (Ze mys L.) seedlings under dmium stress. Journl of Experimentl Biology nd Agriulturl Sienes Apel K nd Hirt H (4). Retive oxygen speies: Metolism, oxidtive stress, nd signl trnsdution. Annul Review of Plnt Biology Arnon DI (1949). Copper enzymes in isolted hloroplsts. Polyphenoloxidse in Bet vulgris. Plnt Physiology Centre for Info Bio Tehnology (CIBTeh) 91

10 Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile Azimi SM, Frni A, Shnnd M nd Lk M (13). Effet of different iofertilizers on seed yield of rley (Hurdeom vulgr L.) Bhmn ultivr. Interntionl Journl of Advned Biologil nd Biomedil Reserh 1(5) Btr NG, Shrm V nd Kumri N (14). Drought-indued hnges in hlorophyll fluoresene, photosyntheti pigments, nd thylkoid memrne proteins of Vign rdite. Journl of Plnt Intertions 9(1) Boominthn R nd Dorn PM (). Ni indued oxidtive stress in roots of the Ni hyper umultor, Alyssum ertolonii. New Phytologist Brnquinho C, Brown DH nd Ctrino F (1997). The ellulr lotion of Cu in lihens nd its effets on memrne integrity nd hlorophyll fluoresene. Environmentl nd Experimentl Botny Bhrdwj D, Ansri MW, Shoo RK nd Tutej N (14). Biofertilizers funtion s key plyer in sustinle griulture y improving soil fertility, plnt tolerne nd rop produtivity. Miroil Cell Ftories Buhnn BB, Gruissem W nd Jones RL (). Biohemistry nd Moleulr Biology of Plnts. (Rokville, MD, USA: Amerin Soiety of Plnt Physiologists). Buege JA nd Aust SD (1978). Mirosoml lipid peroxidtion. Methods in Enzymology Cnolt MY, Bilen S, Çkmkçı R, Ahin F nd Aydın A (6). Effet of plnt growth-promoting teri nd soil omption on rley seedling growth, nutrient uptke, soil properties nd rhizosphere miroflor. Biology nd Fertility of Soils Cohen AC, Bottini R, Pontin M, Berli FJ, Moreno D, Bonlndro H, Trvgli CN nd Pioli PN (15). Azospirillum rsilense meliortes the response of Aridopsis thlin to drought minly vi enhnement of ABA levels. Physiologi Plntrum Delfine S, Tognettir R, Loreto F nd Alvino A (). Physiologil nd growth responses to wter stress in field grown ell pepper (Cpsium nnuum, L.). Journl of Hortiulturl Siene nd Biotehnology 77(6) Deshmuukh PS, Sirm RK nd Shukl DS (1991). Mesurement of ion lekge s sreening tehnique for drought resistne in whet genotypes. Indin Journl of Plnt Physiology Doorenos J nd Pruitt WO (199). Clultion of rop wter requirements. In: Doorenos, J., Pruitt, W.O. (edition), Guideline for Prediting Crop Wter Requirements: FAO Irrigtion nd Dringe. Pper No. 4. (FAO, Rome, Itly) Esmeili M, Heidrzde A nd Rmezni S (16). The effet of plnt growth promoting rhizoteri (PGPR) nd spry of mironutirnt on two soyen (Glyine mx) ultivrs. Journl of Agriulturl nd Biologil Siene 11() Grí-Frile P, Menéndez E nd Rivs R (15). Role of teril iofertilizers in griulture nd forestry. Bioengineering (3) Ginnopolitis CN nd Ries SK (1977). Superoxide dismutse: II. Purifition nd quntittive reltionship with wter-solule protein in seedlings. Plnt Physiology 59() Hrtree EF (197). Determintion of protein: modifition of the Lowry method tht gives liner photometri response. Anlytil Biohemistry Heidri M nd Golpyegni AA (1). Effets of wter stress nd inoultion with plnt growth promoting rhizoteri (PGPR) on ntioxidnt sttus nd photosyntheti pigments in sil (Oimum silium L.). Journl of the Sudi Soiety of Agriulturl Sienes 11(1) Hoglnd DR nd Arnon DI (195). The wter ulture method for growing plnts without soil. Cirulr Cliforni Agriulturl Experiment Sttion 347. Hu XL, Liu RX, Li YH, Wng W, Ti FJ, Xue RL nd Li CH (1). Het shok protein 7 regultes the sisi id-indued ntioxidnt response of mize to omined drought nd het stress. Plnt Growth Regultion Jung S (4). Vrition in ntioxidnt metolism of young nd mture leves of Aridopsis thlin sujeted to drought. Plnt Siene Centre for Info Bio Tehnology (CIBTeh) 9

11 Indin Journl of Plnt Sienes ISSN: (Online) An Open Aess, Online Interntionl Journl Aville t 16 Vol.5 (4) Otoer-Deemer, pp /Aou-Zeid nd Adel-Ltif Reserh Artile Kettner J nd Doerffling K (1995). Biosynthesis nd metolism of sisi id in tomto leves infeted with Botrytis inere. Plnt Journl Khlil SE nd Yousef RMM (14). Study the effet of irrigtion wter regime nd fertilizers on growth, yield nd some fruit qulity of Hiisus sdriff L. Interntionl Journl of Advned Reserh (5) Lihtenthler HK (1987). Chlorophyll nd rotenoids: pigments of photosyntheti iomemrnes. Methods in Enzymolgy Loreto F, Mnnozzi M, Mris C, Nsetti P, Ferrnti F nd Psqulini S (1). Ozone quenhing properties of isoprene nd its ntioxidnt role in leves. Plnt Physiology Mrulnd-Aguirre A, Azon R, Ruiz-Lozno JM nd Aro R (8). Differentil effets of Billus megterium strin on Ltu stiv plnt growth depending on the origin of the rusulr myorrhizl fungus oinoulted: physiologi nd iohemil trits. Journl of Plnt Growth Regultion Mittler R (). Oxidtive stress, ntioxidnts nd stress tolerne. Trends in Plnt Siene Monneveux P nd Belhssen E (1996). The diversity of drought dpttion in the wide. Plnt Growth Regultion Mouss H nd Adel-Aziz SM (8). Comprtive response of drought tolernt nd drought sensitive mize genotypes to wter stress. Austrlin Journl of Crop Siene Noreen Z nd Ashrf M (9). Chnges in ntioxidnt enzymes nd some key metolites in some genetilly diverse ultivrs of rdish (Rphnus stivus L.). Environmentl nd Experimentl Botny 67() Pustovoitov TN, Zhdnov NE nd Zholkevih VN (4). Chnges in the levels of IAA nd ABA in uumer leves under progressive soil drought. Russin Journl of Plnt Physiology Rohmn MM, Begum S, Tlukder MZA, Akhi AH, Amiruzzmn1 M, Ahsn AFMS nd Hossin Z (16). Drought sensitive mize inred shows more oxidtive dmge nd higher ROS svenging enzymes, ut not glyoxlses thn tolernt one t seedling stge. Plnt Omis Journl 9(4) - 3. Sleem M, Arshd M, Hussin S nd Bhtti AS (7). Perspetive of plnt growth promoting rhizoteri (PGPR) ontining ACC deminse in stress griulture. Journl of Industril Miroiology Biotehnology Selvkumr G, Reeth S nd Thmizhiniyn P (1). Response of Biofertilizers on growth, yield ttriutes nd ssoited Protein Profiling hnges of Blkgrm (Vign mungo L.) Hepper. World Applied Sienes Journl 16(1) Sergiev I, Alexiev V nd Krnov E (1997). Effet of spermine, trzine nd omintion etween them on some endogenous protetive systems nd stress mrkers in plnts. Comptes Rendus de l'ademie Bulgre des Siene 51(3) Sokl RR nd Rohlf FJ (1995). Biometry: The Priniples nd Prtie of Sttistis in Biologil Reserh, (W.H. Freemn nd Compny, New York, USA) Treest A (197). Mesurements of Hill retions nd photoredution. In. A Sn Pietro (edition), Methods in Enzymology , (Ademi Press, New York, USA). Turner NC (1981). Tehniques nd experimentl pprohes for the mesurement of plnt wter sttus. Plnt nd Soil Vessey JK (3). Plnt growth promoting Rhizoteri s iofertilizers. Plnt nd Soil Wng C, Yng A, Yin H nd Zhng J (8). Influene of wter stress on endogenous hormone ontents nd ell dmge of mize seedlings. Journl of Integrtive Plnt Biology Xie ZJ, Jing D, Co WX, Di TB nd Jing Q (3). Reltionships of endogenous plnt hormones to umultion of grin protein nd strh in winter whet under different post nthesis soil wter sttuses. Plnt Growth Regultion Zhng H, Jing Y, He Z nd M M (5). Cdmium umultion nd oxidtive urst in grli (Allium stivum). Journl of Plnt Physiology 16(9) Centre for Info Bio Tehnology (CIBTeh) 93