NaCl-PRIMING MITIGATES OXIDATIVE DAMAGE AND Na + ACCUMULATION AND ENHANCES SALT TOLERANCE IN SORGHUM PLANTS

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1 NCl-PRIMING MITIGATES OXIDATIVE DAMAGE AND N + ACCUMULATION AND ENHANCES SALT TOLERANCE IN SORGHUM PLANTS R. S. Mirnd 1, S. O. Pul 2, G. S. Arújo 3, I. N. Vlenç, S. R. N. Mirnd 5, E. Gomes-Filho 6 ABSTRACT: In this study, we tested the hypothesis tht priming with NCl llevites sltindued dmges y upregulting enzymti ntioxidnt system nd ontrolling ioni homeostsis in sorghum plnts. After germintion, uniform Sorghum iolor seedlings were primed with NCl t (ontrol), 1 (P1), 2 (P2) nd 3 mm (P3) (pre-tretment), for seven dys, nd then sujeted to 8 mm NCl-stress. The most striking effets of NCl-priming were registered in plnts from P1-tretment. After five nd ten dys of slinity, lipid peroxidtion nd N + umultion ws found to e drstilly improved in roots nd shoots; however, the inrese ws more severe in non-primed stressed plnts. The lower oxidtive dmge in P1- stressed plnts positively orrelted with higher tivity of tlse (CAT), dismutse superoxide (SOD), sorte peroxidse (APX), guiol peroxidse (G-POD) enzymes under slinity. In generl, CAT nd SOD were the most responsive enzymes to NCl priming, while APX nd G-POD were responsive only fter onset of slinity (five dys) nd/or in single plnt orgn. In onlusion, NCl priming enhnes plnt s pity to retin overumultion of N +, nd limit oxidtive dmge y stimulting effetively ntioxidnt system. KEYWORDS: Antioxidnt enzymes,ioni homeostsis,slt stress. PRÉ-TRATAMENTO COM NCl MINIMIZA OS DANOS OXIDATIVOS E A ACUMULAÇÃO DE N + E AUMENTA A TOLERÂNCIA DE PLANTAS DE SORGO AO ESTRESSE SALINO RESUMO: Neste estudo, foi verifido se o pré-trtmento om NCl livi os efeitos deletérios d slinidde trvés de menismos de ontrole d homeostse iôni e defes ntioxidtiv. Plântuls de Sorghum iolor form ultivds em solução nutritiv ontendo NCl (ontrole), 1 (P1), 2 (P2) e 3 (P3) mm (pré-trtmento), por sete dis. Em 1 Pós-Doutorndo, Depto de Bioquími e Biologi Moleulr, UFC, Fortlez, CE. 2 Doutorndo, Depto de Bioquími e Biologi Moleulr, UFC, CEP 6-55, Fortlez, CE. Fone (85) E-mil: stelmrisop@live.om 3 Doutorndo, Depto de Bioquími e Biologi Moleulr, UFC, Fortlez, CE. Grdundo em Biotenologi, Depto de Bioquími e Biologi Moleulr, UFC, Fortlez, CE. 5 Estgiário, Colégio Estdul Regin Pis, Crteús, CE. 6 Doutor e Professor titulr, Depto de Bioquími e Biologi Moleulr, UFC, Fortlez, CE.

2 R. S. Mirnd et l. seguid, s plnts form sumetids o estresse om NCl 8 mm, sendo nlisds pós 5 e 1 dis de estresse. Os efeitos mis mrntes do pré-trtmento form registrdos ns plnts estressds d ondição P1. A slinidde umentou peroxidção de lipídios e os teores de N + nos órgãos nlisdos; ontudo, os efeitos form miores ns plnts estressds e não prétrtds, em omprção àquels limtds om NCl 1 mm. A redução dos dnos oxidtivos em plnts estressds P1 foi devido à tividde ds enzims tlse (CAT), dismutse do superóxido (SOD), peroxidse do sorto (APX) e peroxidse do guiol (G- POD). As enzims CAT e SOD form positivmente responsivs o pré-trtmento em mos os órgãos e tempos nlisdos, enqunto que s demis enzims form modulds somente pós o estresse slino (5 dis) e/ou em um únio órgão. Conlui-se que limtção de plnts om ixs onentrções de NCl é pz de tivr reposts de tolerâni à slinidde, omo o sistem ntioxidtivo enzimátio e o ontrole d homeostse iôni. PALAVRAS-CHAVE: Enzims ntioxidntes; homeostse iôni; estresse slino. INTRODUCTION In some regions of world the plnt prodution nd rop yields re strongly limited due to ioti stresses inluding soil slinity. The inresed sliniztion proess of griulturl lnd is expeted to hve glol effets, using 3% lnd loss long to the next 25 yers (Wng et l., 23). Thus, efforts re urrently eing mde to selet more slt tolernt genotypes nd/or inrese slt tolerne of rop plnts nd hene hieve sustinle griulture. Plnt priming (pre-tretment of plnts/seeds y previous or onomitnt exposure to stressor or hemil ompounds, mking the plnts more tolernt to future stress events) with some smll moleules hs reeived onsiderle ttention s powerfully tool to indue resistne ginst slt stress. Reent reports hve shown tht hloride sodium (NCl), hydrogen peroxide (H2O2), nitri oxide (NO) nd oligohitosn my t s signling moleule modulting multiple stress-responsive pthwys inresing slt tolerne of numerous plnt rops (Gondim et l., 211; M et l., 212; Pndolfi et l., 216; Gdelh et l., 217). In spite of the importne of plnt priming, there is no informtion regrding the involvement of priming in slt responses of sorghum plnts. Sorghum iolor, n nnul C-grss rop, is not only ommonly onsumed s humn food nd livestok feed, ut it is lso widely employed s suitle feedstok for vriety of iologil proesses, inluding ethnol prodution (Whitfield et l., 212). Sorghum rop is often grown in res of stressful environmentl onditions, suh s drought nd soil slinity. Nevertheless, lthough sorghum hs widely known s modertely slt tolernt rop (Lerd

3 IV INOVAGRI Interntionl Meeting, 217 et l., 23), its ultivtion in severl griulturl res hs eoming serious prolem due to grdul slt umultion in soils. Our working hypothesis ws tht NCl priming mitigtes slt-indued dmges in sorghum plnts y tivting mehnisms for ioni homeostsis ontrol nd ellulr detoxifition. To test this hypothesis, we first exposed sorghum plnts to low levels of NCl nd then sujeted to 8 mm NCl-stress for ten dys. Growth nd iohemil stress inditors were investigted to determine the reltionship etween NCl priming nd slinity tolerne. MATERIAL AND METHODS Seed of sorghum [S. iolor (L.) Moenh] of genotype CSF2 [(otined from Instituto Agronômio de Pernmuo (IPA), Reife, Pernmuo, Brzil] were sown in vermiulite moistened with distilled wter. For dys fter the sowing, the uniform seedlings were trnsferred to plsti trys (1. L) ontining one-hlf strength Hoglnd s nutrient solutions (Hoglnd & Arnon, 195) nd the NCl priming tretments y dding NCl t 1, 2 nd 3 mm to nutrient solution. A plnt group remined in nutrient solution without NCl ddition (no priming). After eight dys of limtion period, the slt tretments were dministered y dding 8 mm NCl in two doses of mm per dy. Nutrient solutions were renewed every three dys nd the hrvests were performed five nd ten dys fter the lst slt ddition. The experiments were rried out in greenhouse, where the middy photosyntheti photon flux density (PPFD) ws 1,3 μmol m -2 s -1, the men ir temperture ws 29. C during the dy nd 26.7ºC t night, nd the min ir reltive humidity ws 65.2%. In eh hrvest time, five plnts from eh tretment were individully hrvested. Firstly, lef re ws evluted through LI-3 lef re meter (LI-COR, In. Linoln, Nersk, USA). Then, plnts were seprted in shoots (leves + stems) nd roots, frozen in liquid nitrogen, nd fter dried y lyophiliztion, the dry mss ws mesured. For ion ontent, 3 mg lyophilized smples from the shoot nd root were homogenized with deionized wter t 5ºC for 1 h. After entrifuged t 3 g for 15 min t room temperture, the superntnt ws olleted nd used to determine K + e N + nd Cl - ontents. The K + nd N + onentrtions were mesured y flme photometry, while the Cl - ontent ws spetrophotometrilly determined ording to Gines et l. (198), sed on the sorne reding t 6 nm with NCl s stndrd. Lipid peroxidtion ws estimted s thiorituri id retive sustnes (TBARS) ording to method of Heth & Pker (1968). The onentrtion of TBARS ws mesured t 532 nm nd the vlue for non-speifi turidity t 6 nm ws sutrted. The TBARS vlues were estimted using its extintion oeffiient of 155 mm -1 m -1.

4 R. S. Mirnd et l. For enzyme ssys, fresh smples were ground in mortr with liquid nitrogen nd then homogenized with extrtion uffer (1 mm potssium phosphte, ph 7., ontining.1 mm EDTA) t ºC. The homogente ws entrifuged t 15, g for 15 min t ºC nd superntnt used for ssys of the tivities of superoxide dismutse (SOD), guiol peroxidse (G-POX), tlse (CAT) nd sorte peroxidse (APX). SOD tivity ws mesured y evluting its ility to inhiit the photohemil redution of nitro lue tetrzolium (NBT), s previously desried y Beuhmp & Friovih (1971). One SOD tivity unit (AU) ws defined s the mount of enzyme required to use 5% inhiition of the NBT photoredution retion. Ctlse nd guiol peroxidse were ssyed ording to the methods of Hvir & MHle (1987) nd Kr & Mishr (1976), y monitoring the sorne t 2 nm nd 2 nm, respetively. CAT nd G-POD tivities were estimted using the molr extintion oeffiient of, respetively, 36 M -1 m -1 nd 26.6 mm -1 m -1. Asorte peroxidse tivity ws mesured y the derese in sorne redings t 29 nm, using the molr extintion oeffiient of 2.8 mm -1 m -1 (Nkno & Asd, 1981). Experimentl design ws ompletely rndomized. For the growth nd ion umultion ssys, the experiment ws divided into five tretments, inluding ontrol (neither NClpriming nor NCl-stress), slt stressed (no NCl-priming nd NCl-stress), P1 (1 mm NClpriming nd NCl-stress), P2 (2 mm-ncl priming nd NCl-stress) nd P3 (3 mm-ncl priming nd NCl-stress). For the lipid peroxidtion nd enzymti ntioxidnt nlyses, the experiment ws rrnged in 2 2 ftoril sheme omposed of two slinity levels ( nd 8 mm NCl) nd two NCl priming tretments [no NCl-priming nd P1 NCl-priming (the level of NCl-priming tht improve slt resistne of S. iolor plnts)]. All nlyses were performed using five plnts (repetitions) per tretment. The dt were sujeted to nlysis of vrine (ANOVA) nd, when differene ws signifint (p.5), the men vlues were ompred using Tukey s test. RESULTS AND DISCUSSION Slt stress is widely known to impir plnt growth nd produtivity y disturing numerous physiologil nd iohemil proesses like ion homeostsis nd ion tivities (Gomes-Filho et l., 28; Mirnd et l., 217). Conordntly, in this study, the dry mss of shoot (DMS), root (DMR) nd totl (DMT) ws drstilly deresed y slinity, irrespetive of NCl priming (Fig. 1); however, the effets were less pronouned in P1-limted plnts. Under 8 mm NCl-stress, P1-limted plnts displyed min vlues of DMS, DMR nd DMT 16% higher thn those of stressed plnts only (Fig. 1A nd 1B). In similr wy, slinity

5 IV INOVAGRI Interntionl Meeting, 217 signifintly redued the lef re (LA) of plnts from ll stress tretments, ut LA vlues of P1-limted plnts were higher thn those of stressed (Fig. 1C). Our dt lerly evidene tht NCl-priming ws effetive to redue slt deleterious effets on growth of sorghum plnts (Fig. 1), whih ws losely relted to etter ioni homeostsis. Herein, slinity promoted severe redutions in K + ontent nd n over umultion of N + in oth shoot nd root tissues of sorghum plnts (Fig. 1A nd 1B). In shoot, under slt stress, the lowest N + umultion ws oserved in P1-limted plnts (Fig. 1A). Yet, in root, the N + ontent ws higher in plnts from P3 tretment, followed y those from other stress nd NCl-priming tretments (Fig. 1B). As onsequene, lthough slt stress signifintly deresed the K + /N + rtio in oth plnt tissues, greter K + /N + rtio ws estlished in the shoot of P1-limted plnts in omprison with other slt tretments (Fig. 2A nd 2B). Consistently, low N + umultion together with elevted K + /N + rtio hs een onsidered s n importnt mehnism for slt tolerne in plnt speies (Ymguhi et l., 213; Mirnd et l., 217). In ft, our findings demonstrted tht the most slt tolernt sorghum plnts (P1-limted) showed lower ontent of toxi N + ions oupled to greter K + /N + rtio in leves (Fig. 2A nd 2C). In order to evlute if NCl-priming llevites slt-indued oxidtive dmge, the lipid peroxidtion nd enzymti ntioxidnts were mesured. For ll nlyzed time-points (5 nd 1 dys of slt tretments), slt stress drmtilly inresed the lipid peroxidtion in nonlimted plnts, it eing more ggressive in root tissues. On the other hnd, slight inrese in lipid peroxidtion y slinity in P1-limted plnts ws oserved only fter ten dys of slt tretment (Fig. 3). In ddition, under slinity, P1-limted plnts displyed vlues of lipid peroxidtion lower thn those of non-limted plnts, espeilly in photosyntheti tissues. These dt suggest tht slinity ould use injuries to ellulr omponents like lipids, proteins nd nulei ids, s previously reported in Jtroph urs (Gdelh et l., 217) nd other plnt speies (Demidhik, 215), ut NCl-priming n prtilly prevent the oxidtive dmge normlly used y slt stress. To void the slt-indued oxidtive dmge, plnt ells hve developed n elegnt defense system, inluding enzymti nd non-enzymti omponents. Reent reports hve shown tht plnt speies employing highly effiient ntioxidnt systems hd improved slt stress tolerne (Notor & Foyer, 216; Gdelh et l., 217). This ide is supported y the fts tht ntioxidnt enzyme tivities signifintly inresed in P1-limted stressed sorghum plnts, wheres the lipid peroxidtion signifintly deresed in omprison to nonlimted stressed plnts (Fig. 3 nd ). Under ontrol onditions, CAT, SOD nd G-POD tivity of P1-limted plnts ws igger thn non-limted ones, in oth plnt orgns (Fig. ). In generl, under slt stress,

6 R. S. Mirnd et l. CAT, SOD, G-POD nd APX tivities were inresed in roots nd shoots of plnts from ll tretments; nevertheless, P1-limted stressed plnts displyed higher CAT, SOD, G-POD nd APX tivity vlues thn those of non-limted stressed plnts, exept for APX in roots nd G-POD in shoot t the seond time-point. Our dt demonstrted tht, when sorghum plnts were limted with 1 mm NCl, higher opertion of enzymti ntioxidnts under 8 mm- NCl stressis oserved. CONCLUSION Our findings revel tht priming of seedlings with NCl triggers importnt responses ginst slt stress nd improves plnt s slt tolerne of S. iolor. The role of NCl priming to overome slinity hrmful effets seems to rely prtly on its prtiulr properties to indue mehnisms for regulting ellulr ion homeostsis nd ntioxidnt omponents therey lleviting oxidtive dmge. Thus, NCl priming emerges s plusile ultivtion tehnique to prevent severe growth losses due to slinity nd might hve signifint prtil pplition for ultivting S. iolor plnts in sline environments. ACKNOWLEDGMENTS We re very grteful to finnil support provided y the Conselho Nionl de Desenvolvimento Científio e Tenológio (CNPq), Instituto Nionl de Ciêni e Tenologi em Slinidde (INCTSl) nd Coordenção de Aperfeiçomento de Pessol de Nível Superior (CAPES). REFERENCES BEAUCHAMP, C.; FRICOVICH, I. Superoxide dismutse: improved ssys nd n ssy pplile to rylmide gels. Anlytil Biohemistry, v., p , DEMIDCHIK, V. Mehnisms of oxidtive stress in plnts: from lssil hemistry to ell iology. Environmentl nd Experimentl Botny, v.19, p , 215. GADELHA, C.G.; MIRANDA, R.S.; ALENCAR, N.L.M.; COSTA, J.H.; PRISCO, J.T.; GOMES-FILHO, E. Exogenous nitri oxide improves slt tolerne during estlishment of Jtrophurs seedlings y meliorting oxidtive dmge nd toxi ion umultion. Journl of Plnt Physiology, v.212, p.69-79, 217. GAINES, T.P.; PARKER, M.B.; GASCHO, G.J. Automted determintion of hlorides in soil nd plnt tissue y sodium nitrte. Agronomy Journl, v.76, p , 198.

7 IV INOVAGRI Interntionl Meeting, 217 GOMES-FILHO, E.; LIMA, C.R.F.M.; COSTA, J.H.; SILVA, A.C.M.; LIMA, M.G.S.; LACERDA, C.F.; PRISCO, J.T. Cowpe rionulese: properties nd effet of NCl-slinity on its tivtion during seed germintion nd seedling estlishment. Plnt Cell Reports, v.27, p , 28. GONDIM, F.A.; GOMES-FILHO, E.; COSTA, J.H.; MENDES ALENCAR, N.L.; PRISCO, J.T. Ctlse plys key role in slt stress limtion indued y hydrogen peroxide pretretment in mize. Plnt Physiology nd Biohemistry, v.56, p.62-71, 212. HAVIR, E.; MCHALE, N.A. Biohemil nd developmentl hrteriztion of multiple forms of tlses in too leves.plnt Physiology, v.8, p.5-55, HEATH, R.L.; PACKER, L. Photoperoxidtion in isolted hloroplsts. I. Kinetis nd stoihiometry of ftty id peroxidtion. Arhives of Biohemistry nd Biophysis, v.125, p , HOAGLAND, D.R.; ARNON, D.I.The wter ulture method for growing plnts without soil. Cliforni Agriulturl Experiment Sttion, v.37, p.1-39, 195. KAR, M.; MISHRA, D. Ctlse, peroxidse, nd polyphenoloxidse tivities during rie lef senesene.plnt Physiology, v.57, p , LACERDA, C.F.; CAMBRAIA, J.; OLIVA, M.A.; RUIZ, H.A. Osmoti djustment in roots nd leves of two sorghum genotypes under NCl stress. Brzilin Journl of Plnt Physiology, v.15, p , 23. MA, L.; LI, Y.; YU, C.; WANG, Y.; LI, X.; LI, N.; CHEN, Q.; BU, N. Allevition of exogenous oligohitosn on whet seedlings growth under slt stress. Protoplsm, v.29, p , 212. MIRANDA, R.S.; MESQUITA, R.O.; COSTA, J.H.; ALVAREZ-PIZARRO, J.C.; PRISCO, J.T.; GOMES-FILHO, E. Integrtive ontrol etween proton pumps nd SOS1 ntiporters in roots is ruil for mintining low N + umultion nd slt tolerne in mmonium-supplied Sorghumiolor. Plnt nd Cell Physiology, v.58, p , 217. NAKANO, Y.; ASADA, K. Hydrogen peroxide is svenged y sorte-speifi peroxidse in spinsh hloroplsts. Plnt nd Cell Physiology, v.22, p , NOCTOR, G.; FOYER, C.H. Intrellulr redox omprtmenttion nd ROS-relted ommunition in regultion nd signling.plnt Physiology, v.171, p , 216. PANDOLFI, C.; AZZARELLO, E.; MANCUSO, S.; SHABALA, S. Alimtion improves slt stress tolerne in Ze mys plnts. Journl of Plnt Physiology, v.21, p.1-8, 216. WANG, W.; VINOCUR, B.; ALTMAN, A. Plnt responses to drought, slinity nd extreme tempertures: towrds geneti engineering for stress tolerne. Plnt, v.218, p.1-1, 23. WHITFIELD, M. B.; CHINN, M. S.; VEAL, M. W. Proessing of mterils derived from sweet sorghum for iosed produts. Industril Crops nd Produts, v.37, p , 212. YAMAGUCHI, T.; HAMAMOTO, S.; UOZUMI, N. Sodium trnsport system in plnt ells. Frontiers in Plnt Siene, v., p.1-7, 213.

8 R. S. Mirnd et l. Shoot nd root dry mss (g/plnt) Totl dry mss (g/plnt) Root d d Shoot e e (A) (B) Figure 1. Shoot nd root (A) nd totl (B) dry mss, nd lef re (C) of Sorghum iolor plnts in sene (ontrol) nd presene of 8 mm-ncl stress (Stress, P1, P2 nd P3). The plnts were primed with NCl t (stress, negtive ontrol), 1 (P1), 2 (P2) nd 3 mm for eight dys prior to imposition of slt tretments. The mesurements were otined t ten dys fter slinity imposition. Vlues represent the mens of five repetitions + stndrd error. Different lowerse letters represent signifint differenes due to slt stress using Tukey s test (p.5). 75 (C) Lef re (m 2 /plnt) d e K + nd N + ( mol g -1 DM) Control Stress P1 P2 P3 N + Shoot K + (A) Root N + K + (B) K + nd N + ( mol g -1 DM) K + /N + rtio (C) (D) K + /N + rtio. Control Stress P1 P2 P3 Control Stress P1 P2 P3 Figure 2. Conentrtions of potssium (K + ) nd sodium (N + ) (A,B) nd K + /N + rtio (C,D) in shoot nd roots of Sorghum iolor plnts in sene (ontrol) nd presene of 8 mm-ncl stress (Stress, P1, P2 nd P3). More detils s Fig. 1.

9 IV INOVAGRI Interntionl Meeting, 217 Lipid peroxidtion (nmol g -1 FM) Lipid peroxidtion (nmol g -1 FM) Control P1/Control B B B Slt stressed P1/Slt stressed B B A B A B (A) (B) Figure 3. Lipid peroxidtion in shoot (A) nd root (B) tissues of Sorghum iolor plnts in sene (ontrol) nd presene of 8 mm NCl-stress (slt stressed) nd primed with NCl t nd 1 mm. Vlues re given s the men of five iologil repetitions + stndrd error. At eh time point, signifint differenes due to NCl-priming t the sme slt level (ontrol P1/ontrol or slt stressed P1/slt stressed) re denoted y different pitl letters, wheres signifint differenes due to slt stress in the sme NCl-priming (ontrol slt stressed or P1/ontrol P1/slt stressed) re indited y different lowerse letters, using Tukey s test (p.5). Control Stress Control Stress 5 d 1 d

10 R. S. Mirnd et l. Shoot Root Ctlse Control A B B P1/Control B B A (A) Slt stressed B A B P1/Slt stressed A B B (B) Ctlse Superoxide dismutse (AU A A B A B (C) A A B A A B (D) Superoxide dismutse (AU Asorte peroxidse Guiol peroxidse B B Control Stress A B A A B A B B Control Stress (E) (G) B A B B (F) (H) B A A B A B Control Stress Control Stress Asorte peroxidse Guiol peroxidse 5 d 1 d 5 d 1 d Figure. Ctlse (A, B), superoxide dismutse (C, D), sorte peroxidse (E, F) nd guiol peroxidse (G, H) tivity in shoot nd roots of Sorghum iolor plnts in sene (ontrol) nd presene of 8 mm NCl-stress (slt stressed) nd primed with NCl t nd 1 mm. More detils s Fig. 3.