Alleviation of oxidative stress induced by drought stress through priming by β- aminobutyric acid (BABA) in Rapeseed (Brassica napus L.

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1 223 Allevition of oxidtive stress indued y drought stress through priming y β- minoutyri id (BABA) in Rpeseed (Brssi npus L.) plnts Ned Mohmdi 1,2, Amin Bghizdeh 3*, Sr Sdtmnd 1, Zhr Asrr 4 1. Deprtment of Plnt Biology, Fulty of Bsi Siene, Siene nd Reserh Brnh, Islmi Azd University, Tehrn, Irn 2. Young Reserhers nd Elites Clu, Kermn Brnh, Islmi Azd University, Kermn, Irn. 3.Deprtment of Biotehnology, Institute of Siene nd High Tehnology nd Environmentl Sienes, Grdute University of Advned Tehnology, Kermn-Irn 4.Biology Deprtment, Fulty of Siene, Shhid Bhonr University of Kermn, Kermn, Irn. Astrt The ojetive of this study ws to investigte the role of β-minoutyri id (BABA) (, 3µM) in redution of oxidtive dmges in leves nd roots of Brssi npus L. under drought stress (, -.2, -.4 MP). β- minoutyri id ws investigted s n internl regultor hormone nd its role in defense mehnisms ginst ioti nd ioti stresses. In this study, BABA pretretment prevented drought indued derese in K + ontent nd inrese in lipid peroxidtion nd H 2O 2 ontent. Mild nd severe drought stress inresed lef nd root MDA nd other ldehydes ontent. In BABA pretreted plnts under stress, lef nd root MDA nd other ldehydes ontent signifintly deresed s ompre to drought ondition. In ddition, in drought stressed plnts non-enzymti ntioxidnts [GSH] were elevted over the ontrol nd BABA pretretment inresed GSH ontent. The N + nd C ++ onentrtion inresed under mild nd severe drought stress. On the sis of the results, BABA-treted plnts exhiited enhned drought tolerne. Keywords: non-enzymti ntioxidnts, β-minoutyri id, drought stress, oxidtive stress, Brssi npus. Mohmdi, N., A. Bghizdeh, S. Sdtmnd, Z. Asrr.217. 'Allevition of oxidtive stress indued y drought stress through priming y β-minoutyri id (BABA) in Rpeseed (Brssi npus L.) plnts'. Irnin Journl of Plnt Physiology 7 (4), Introdution Wter stress due to drought n led to mjor physiologil nd iohemil disruptions suh s redued photosynthesis (Lowlor nd Corni, 22) nd mrked hnges in gene expression (Romo et l. 21). *Corresponding uthor E-mil ddress: min_4156@yhoo.om Reeived: Mrh, 216 Aepted: My, 217 In humid nd temperture res, drought periods re mjor ftor impeding plnt growth nd development. Plnts hve different mehnisms to void wter defiit. One of these responses is prodution of sisi id (ABA) tht, in turn, elevtes ytosoli C 2+ onentrtions in gurd ells leding to stomtl losure. Closure of stomt s result of wter defiit nd onsequent derese in CO 2 onentrtion in the lef mesophyll results in the umultion of NADPH in the hloroplsts under suh onditions, where

2 224 Irnin Journl of Plnt Physiology, Vol (7), No (4) NADP is limiting, O 2 ts s n lterntive eletron eptor resulting in the formtion of superoxide rdil (Bisk 1994). The O 2, through series of univlent redution retion, produes hydrogen peroxide (H 2O 2) hydroxyl rdil (HO ) nd singlet oxygen ( O 2). This retive oxygen speies (ROS) re highly toxi nd n dmge importnt ellulr iomoleules suh s lipids, proteins, nulei ids nd hlorophyll (Bisk 1994). Mintining high level of enzymti ntioxidnts suh s tlse (CAT), peroxidse (POD) nd other ompounds suh s Asorte (As), Mlondildehyde (MDA), α-tooferol nd rotenoids tivity my ontriute to drought tolerne y inresing the pity etter protetion mehnism ginst oxidtive dmge (Shrm nd duy 25). Intrellulr homeostsis of sodium nd potssium for ytosol enzyme tion, mintenne of memrne potentil nd regultion of ell volume. Under slt stress, mintenne of K + nd N + homeostsis is even more severe, so regultion of ion trnsfer y signs of slinity stress provides model for understnding the omplete regultion of ion homeostsis in plnt ells. In ddition to understnding how plnts ope with exess N + in the environment, in terms of griulture is importnt. N + stress is disrupted K + uptke y the root ells, when N + enters the ell, it umultes in high levels nd will e toxi for plnt. To prevent of growth, stop or ell deth, exess N + should e omitted or enter the vuoles (hsegv et l 2). A ommon theme underlying responses to rrnge of ioti nd ioti stresses is the phenomenon of priming (Wu et l. 21). Priming refers to phenomenon where plnts re sensitized to stress (Conrth et l. 22; Vn der Ent et l. 29). Over the pst yers, priminginduing tivity in plnts hs een reported for mny nturl nd syntheti ompounds tht inlude the non-protein mino id β-mino utyri id (BABA) (Bekers 27). BABA is non-protein mino id whih ours rrely in nture. The only report in onnetion to plnts desries its presene in root exudtes of tomto plnts grown in solrized soil (J et l. 21). It ws shown tht, the BABA inreses Aridopsis resistne to different, unrelted stress suh s miroil pthogens, slt, drought nd het shok (Zimerly et l. 2; 21; 28; Ton nd muh-mni 24; J et l. 25). Reently Co et l (29) showed tht BABA enhned hevy metl dmium resistne through glutthione-dependent pthwy in Aridopsis. BABA ts through potentition of ABA-dependent signling pthwy nd sliyli id dependent defene mehnism (Zimerly et l. 2). However, little is known out the role of BABA in ntioxidnt metolisms under drought stress. Therefore, the ojetive of this study ws to investigte the umultion of ROS nd ntioxidnt system nd Chnges in ion levels in leves nd roots of Brssi npus L. under drought stress nd fter priming y BABA. Mterils nd Methods Plnt mteril nd tretment Seeds of Brssi npus L. Cv. Mdonn were olleted from Krj Agriulturl Centre. Seeds were sterilized using.1% sodium hypohlorite solution, wshed with distilled wter nd plnted in pot filled with perlite. Pots were trnsferred to growth hmer with dy/night temperture of 25/2 C nd 16 h light/8 h drk photoperiod, with reltive humidity of 7%. During the first week of seed sowing, seedlings were irrigted with distilled wter. For the rest of experiment, hlf strength Hoglnd nutrient solution ws used to irrigte plnts every other dy. Drought ws indued y stopping to wter 4 week-old plnts 1 dy fter BABA (Fluk) (, 3µM) tretment y soil drenh. Three level of soil moisture were pplied. (1) Well-wtered (C): the soil wter potentil ws ontrolled t filed pity. (2) Mild drought stress (D1): the soil wter potentil ws -.2MP. (3) Severe drought stress (D2): the soil wter potentil ws -.4Mp. At the end of experiment, the leves nd roots of plnts were hrvested nd immeditely were frozen in liquid nitrogen nd stored t -8ºC for the future nlysis. Lipid peroxidtion

3 β-minoutyri id (BABA) effets in Brssi npus under stress onditions 225 For eh smple,.2 g of the lef tissue ws homogenized in 1 ml of.1% (w/v) trihloroeti id (TCA). The homogente ws entrifuged t 1 g for 5 min. For every 1ml of liquot, 4ml of 2% TCA ontining.5% thioriturie id ws dded. Mixture ws heted t 9 C for 3 min. Smples were ooled on ie for 5 min nd then re-entrifuged t 1 g for 1 min nd sorne of the superntnt ws reorded t 532 nd 6nm. For MDA mesurement, the non speifi sorne of superntnt t 6 nm ws sutrted from the mximum sorne t 532nm nd n extintion oeffiient of / M Cm nd ws used for determintion of MDA onentrtion (Heth nd Pker, 1969). For other ldehydes, n extintion oeffiient (ε) of / M Cm ws used t 455 nm t the verge of e otined for other ldehydes (propnl, utnl, hexnl, heptnl nd propnlheptnl nd propnl dimethyletl). Results were expressed s nm g -1 FW. Hydrogen peroxide ontent H 2O 2 ontent ws mesured spetrophotometrilly fter retion with potssium iodide (KI) ording to the method of (Alexiev et l. 21). Lef tissues (5 mg) were homogenized in ie th with5 ml.1% TCA. The homogente ws entrifuged t 12 g for 15min. The retion mixture onsisted of.5 ml of superntnt,.5 ml of 1mM potssium phosphte uffer (ph 7.) nd 2 ml regent (1M KI in fresh doule-distilled wter). The lnk proe onsisted of.1% TCA in the sene of lef extrt. The retion ws rried out for 1 h in drkness nd sorne ws mesured t 39 nm. The mount of hydrogen peroxide ws lulted using stndrd urve prepred with known onentrtions of H 2O 2. Minerl ontent K +, N + nd C 2+ ontents were determined using n tomi sorption spetrophotometer (Shimdzu, model 61, Jpn) fter wet digestion of the sh with nitri id nd expressed s mg g 1 DW. Determintion of totl glutthione The level of totl id-solule SH ompound (glutthione GSH) ws determined with Ellmn s regent (1959). Smples of.5 g were homogenized in 15% m-phosphori id. The homogente ws entrifuged t 1 g for 3min. The retion mixture onsisted of 2 µl of superntnt, 2.6 ml of 1mM sodium phosphte uffer (ph 7.7) nd 2 µl DTNB (6 M). The sorne t 412 nm ws mesured fter 3 min. The GSH onentrtion ws determined from stndrd urve. Sttistil nlysis Dt for eh prmeter were sujeted to ANOVA nd signifint differenes etween tretment mens were determined y Dunn Multiple Rnge test using the SPSS softwre. Dt re shown s mens with three replites nd signifine ws determined t the 95% onfidene (p.5) limits. Tle 1. Effets of BABA (,3 µm) nd drought stress (, -.2,-.4 MP) on H2O2, MDA nd Other ldehydes ontent in the leves nd root of B. npus. Tretment H 2O 2 (µmg -1 FW)/lef H2O2 (µm g -1 FW)root MDA(nMg -1 FW) lef MDA(nMg -1 FW) root Other ldehydes (nmg -1 FW) lef Other ldehydes (nmg-1 FW) root Control 22.2±2.1 d 4.47±.2 d.66±.37 d 131.7± ±.3 e 1.9±.76 D1 67.3± ± ± ± ± ±.37 D1+BABA 44.6± ±.22 e 1.4±.33 d 136.9± ±.12 d 2.3±.25 D ± ± ± ± ± ±.7 D2+BABA 6.9±2 5.2± ± ± ± ±.216

4 N + Content (mg g -1 DW) K+ Content ( g -1 DW) 226 Irnin Journl of Plnt Physiology, Vol (7), No (4) Lef Root Fig. I. Effets of mild nd severe drought stress nd pretretment of BABA on K + ontent in B. npus under drought stress Lef Root Fig.II. Effets of mild nd severe drought stress nd pretretment of BABA on N+ ontent in B. npus under drought stress. Results The lef H 2O 2 ontent inresed 3-fold nd 6.3-fold under mild nd severe drought stress, respetively, reltive to the ontrol. However, BABA-treted plnts showed signifint redution in H 2O 2 ontent to 66% nd 43% s ompre to non-treted plnts under mild nd severe drought stress (Tle 1). Mild nd severe drought stress inresed 1.8-fold nd 3.5-fold root H 2O 2 ontent. In BABA pre-treted plnts under stress, root H 2O 2

5 GSH Content (µm g -1 FW) C2+ Content (mgg -1 DW) β-minoutyri id (BABA) effets in Brssi npus under stress onditions 227 ontent signifintly deresed to 41% nd 34% s e d d 4 2 Lef Root Fig. III. Effets of mild nd severe drought stress nd pretretment of BABA on C2+ ontent in B. npus under drought stress d d.2.1 Lef Root Fig.IV. Effets of mild nd severe drought stress nd pretretment of BABA on GSH ontent in B. npus under drought stress.

6 228 Irnin Journl of Plnt Physiology, Vol (7), No (4) ompre to drought ondition (Tle 1). Mild nd severe drought stress inresed 2.7-fold nd 3.6- fold lef MDA ontent, 1.12fold nd 1.56-fold root MDA ontent, 4-fold nd 9-fold lef other ldehydes ontent, 1.6-fold nd 2.7-fold root other ldehydes ontent. In BABA pre-treted plnts under stress, lef nd root MDA nd other ldehydes ontent signifintly deresed to 57.7% nd 58.3% for lef MDA, 92% nd 71% for root MDA, 69% nd 7.6% for lef other ldehydes nd 75%, 74% for root other ldehydes ontent s ompre to drought ondition (Tle 1). As shown in Fig. I, K + ontent ws signifintly deresed to 11%, 25.4% for lef nd 17.4%, 55% for root under mild nd severe drought stress respetively. A signifint inresent K + ontent out 1.2-fold for root nd 1.1-fold for lef BABA-treted plnts thn those of wter treted plnts under stress. The lef N + onentrtion inresed to 1.3-fold nd 1.5-fold under mild nd severe drought stress, respetively. Root N + onentrtion signifintly inresed to 1.1-fold nd 1.3-fold in mild nd severe drought stress. In ontrst N + ontent ws not signifintly ffeted y BABA (Fig. II). Mild nd severe drought stress used signifint inrese in C 2+ ontent to 1.3-fold, 1.5- fold in lef nd 1.1-fold, 1.45-fold in root respetively. BABA inresed the C 2+ ontent, up to 1.16-fold, fold reltive to mild nd severe drought ondition (Fig. III). The lef GSH ontent inresed signifintly 1.4-fold nd 1.7-fold nd the root GSH ontent inresed 1.2-fold, 1.4-fold under mild nd severe drought stress reltive to ontrol (Fig. IV). As shown in Fig. IV, GSH ontent inresed y BABA pretretment in plnts under drought ondition. Disussion On the sis of the results, BABA-treted plnts exhiited enhned drought tolerne. J hve shown tht pretretment of Aridopsis plnts with BABA, redued the wilting rte y 5% (J et l. 25). The ion lekge is n inditor of ell memrne stility nd integrity, whih is ommonly onsidered s one of the est physiologil omponents of drought tolerne (Liu et l. 211). The inresing of MDA nd other ldehydes, index of lipid peroxidtion, ws ompnied y n inrese of ion lekge in drought ondition. Our finding illustrted tht, BABA led to redution in lipid peroxidtion. Mintining the integrity of ellulr memrne under stress is onsidered prt of drought tolerne mehnisms (Korkmz 27). K + is mjor tion in ell orgniztion nd it ws reported to e mjor ontriutor to osmoti djustment under stress ondition in severl speies (Sntos-Diz nd Alejo-Oho 1994). Redution in K + nd inrese in N + hs lso een reported in whet plnt under drought ondition (Munns nd King 1988). Co et l found tht higher K + ontent ws deteted in shoot nd root of BABA-treted seedlings thn those of wter treted seedlings. They showed tht the trnsript level of AtHAK5 nd LKS1 involved in K + uptke in BABA-treted plnts ws signifint higher thn tht in wter treted. This result suggest tht BABA ply role in inresing K + uptke, t lest in prt, vi modultion of AtHAK5 nd LKS1 (Co et l. 28). The umultion of O 2 - ourred in the leves did not go long with the hnges in totl SOD tivity (Crvlho 28). The inresed APX in the leves nd inrese GR tivity in roots nd unhnged APX tivity in the root of plnts my help mintin levels of ASA nd GSH (Foyer 1993). Soil drenh tretment with the hemil BABA primes pproprite defene mehnisms nd provides long-term protetion ginst iotrophi teri (Zimerly 2), nerotrophi fungi (Zimmerli et l. 21) nd ioti stresses (J et l., 25). Zhng (2) suggests tht BABA is le to indue drought tolerne in Aridopsis plnt dependent of funtionl ABA signlling. The erlier nd fster ABA prodution, stomtl losure nd expression of ABA-regulted genes led to n enhned wter use effiieny of the plnts (J et l. 25). Pre-tretment with BABA redued the level of lipid peroxidtion nd enhned the survivl in Brssi npus exposed to drought stress. Priming is usully defined s sensitiztion to stress responsiveness. As result, priming oosts the plnt s defene response nd primed plnts re more resistne to ioti nd ioti stress. In Wu et l. study ws shown tht BABA ts s hemil stress nd therefore indues the stress-indued morphogeni response (SIMR) in Aridopsis.

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