Stomatal behavior and components of the antioxidative system in coffee plants under water stress

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1 Antioxidtive system in offee plnts under wter stress 77 Stomtl ehvior nd omponents of the ntioxidtive system in offee plnts under wter stress Sidnei Deuner 1 ; José Donizeti Alves 2 *; Ilisndr Znndre 3 ; Ptríi de Fátim Pereir Goulrt 4 ; Neidiquele Mri Silveir 2 ; Pôl de Cstro Henrique 2 ; Alessndro Crlos Mesquit 5 1 UFPel Depto. de Botâni, Instituto de Biologi, C.P Pelots, RS Brsil. 2 UFLA Depto. de Biologi, Setor de Fisiologi Vegetl, C.P Lvrs, MG Brsil. 3 Emrp Clim Temperdo, C.P Pelots, RS Brsil. 4 UNILAVRAS Centro Universitário de Lvrs, 372- Lvrs, MG Brsil 5 Emrp Cfé, C.P Lvrs, MG Brsil *Corresponding uthor <jdlves@di.ufl.r> ABSTRACT: Coffee (Coffe ri) plnts show positive reltionship etween stomtl losure nd formtion nd umultion of H 2. However, for offee plnts under wter restrition suh reltionship hs never een studied. The ojetive of the present study ws evlute the stomtl movement nd the ntioxidnt pity of offee seedlings under different wter regimes. Eight months old offee seedlings of v. Ctuí IAC 99 were sumitted to field pity, grdul nd totl suspension of irrigtion during period of 21 dys. Evlutions of lef wter potentil (Ψ w ) were performed in the eginning of the morning, nd stomtl resistne, trnspirtion rte nd vpor pressure defiit were determined t 1 m nd 5 pm. All iohemil nd enzymti determintions were performed in leves olleted t 5 pm. Evlutions nd smplings were performed t three dys intervls. There ws no vrition in Ψ w during the evluted period for plnts in field pity. However, n expressive derese of Ψ w following dy 12, rehing vlues ner - MP t the end of the experiment ws oserved for plnts sumitted to grdul suspension of irrigtion. For plnts sumitted to totl suspension of irrigtion, Ψ w dereses fter the sixth dy, rehing - MP t dy 15. The dey of Ψ w in plnts sumitted to grdul nd totl suspension of irrigtion refleted in inresed stomtl resistne nd in deresed trnspirtion rte leding to n inrese in hydrogen peroxide formtion nd, on finl stges, inrese in lipid peroxidtion. As onlusion, n inrese in the tivity of ntioxidnt enzymes s well s in the levels of sorte nd dehydrosorte ws oserved, whih t in the detoxifition of free rdils formed s result of the wter stress. Key words: Coffe ri, oxidtive stress, ntioxidnts, wter stress Comportmento estomátio e omponentes do sistem ntioxidnte em feeiros so estresse hídrio RESUMO: Pr o feeiro (Coffe ri) existe um omprovd relção positiv entre fehmento estomátio e formção e úmulo de H 2. Entretnto, tl relção pr ultur so restrição hídri ind não foi estudd. Avliou-se o movimento estomátio e pidde ntioxidnte em muds de feeiro so diferentes regimes hídrios. Muds de feeiro v. Ctuí IAC 99, om oito meses de idde, form sumetids à pidde de mpo, suspensão grdtiv e suspensão totl d irrigção por um período de 21 dis. Form relizds vlições do potenil hídrio (Ψ w ) folir n ntemnhã e resistêni estomáti, tx trnspirtóri e défiit de pressão de vpor form vlidos s 1h e 17h. As determinções ioquímis e enzimátis form relizds em folhs oletds às 17h. Tods s vlições e olets form relizds em intervlos de três dis. Ns plnts em pidde de mpo não houve vrição no Ψ w durnte o período de vlição. Pr suspensão grdtiv d irrigção, houve qued expressiv prtir dos 12 dis, hegndo próximo -2,5 Mp, o finl do experimento. Já ns plnts em suspensão totl d irrigção oservou-se qued no Ψ w prtir do sexto di, hegndo -2,5 MP os 15 dis. A qued no Ψ w pr s plnts em suspensão grdul e totl d irrigção refletiu em umentos n resistêni estomáti e diminuição d tx trnspirtóri, osionndo umento n formção de peróxido de hidrogênio e nos períodos finis, umentos n peroxidção de lipídios. Em onseqüêni oervrm-se umentos n tividde ds enzims ntioxidntes, em omo nos teores de sorto e dehidrosorto, tundo n detoxifição dos rdiis livres formdos em função do estresse. Plvrs-hve: Coffe ri, estresse oxidtivo, ntioxidntes, estresse hídrio Introdution Brzil is the min ountry exporter of offee (Coffe ri) nd it is responsile for one third of the world prodution (Pereir et l., 27). Brzilin prodution ould e even more signifint if unfvorle environmentl onditions, suh s drought, did not our frequently (Gomes et l., 28). Prtiulrly for offee trees, Si. Agri. (Piri, Brz.), v.68, n.1, p.77-85, Jnury/Ferury 211

2 78 Deuner et l. dereses in the wter supply imposes sustntil redution in plnt growth (Oliveir et l., 22; Freits et l., 27), lthough no fetures of wter defiieny n e seen (Grisi et l., 28). In these ses, rpid stomtl losure in leves ours in response to wter defiieny (Nsimento et l., 28) nd optimizes wter use effiieny, therey plying ruil roles in drought stress tolerne. When plnts re drought-stressed, sisi id (ABA) indues stomtl losure y reduing the turgor of gurd ells under wter defiit (An et l., 28). By opening nd losing stomt, the gurd ells ontrol trnspirtion to regulte wter loss or retention. ABA nd H 2 re produed nd umulted in plnts under dverse environmentl onditions suh s drought, nd re ruil in signling dptive responses, inluding stomtl losure (Zhng et l., 21) nd ntioxidnt defense (Zhng et l., 26). Zhng et l. (21) reported the umultion of H 2 in the gurd-ells under wter defiit, tivted y n inrese in the ABA prodution. These uthors lerly show tht H 2 is n essentil signl in ABA-indued stomtl losure. Environmentl stresses promote enhned prodution retive oxygen speies (ROS), suh s superoxide nion ( - ), hydrogen peroxide (H 2 ), nd mny free rdils nd its onsequent umultion in plnts nd therey dmge DNA, proteins nd lipids (Grtão et l., 25; Vsonelos et l., 29). To protet its ells from ROS, drought tolernt plnts tivte n oxidtive defense system, formed y enzymti nd non-enzymti omponents ple of mintining fvorle lne etween ROS nd the detoxifying pity (Pompeu et l., 28; Vsonelos et l., 29). Moreover, plnt responses to different types of stresses re ssoited with genertion of ROS, suggesting tht ROS my funtion s ommon signl in signling pthwys of plnt stress responses (Grtão et l., 25). Coffee studies re onsolidted in the sense tht there is positive reltionship etween stomtl losure nd formtion nd umultion of H 2 (Deuner et l., 28). In this previous report we evluted the exogenous pplition of sori id nd hydrogen peroxide on enzyme tivity of the ntioxidnt system nd its reltion to stomtl opening. Applition of H 2 redued the stomtl ondutne nd onsequently the trnspirtion rte ompred to the ontrol plnts, nd t the sme time, promoted higher ntioxidnt tivity of the enzymes. When plnts were spryed with sori id, higher stomtl ondutne nd trnspirtion rte were found in the first hour of evlution. Coffee under wter restrition hs the level of H 2 inresed nd triggers mehnisms relted to stomtl losure nd detoxifition of H 2 in order to retin its degree of hydrtion. These findings provide n interesting insight into the mehnism of stomt-regulted drought stress tolerne in offee trees nd importnt ontriutions to etter understnding of the mngement of this importnt rop offee under wter stress. Therefore, this pper imed to evlute the stomtl movement nd the ntioxidnt pity of offee seedlings under different wter regimes. Mteril nd Methods Eight months old seedlings of Coffe ri, ultivr Ctuí IAC 99, were grown in 3L plsti gs ontining s sustrte mixture of soil nd ttle mnure. The plnts remined for two months in greenhouse, overed with lk-film tht interepts 3% solr rdition. During this period, the plnts were wtered regulrly, mintining the wter level within the field pity until the tretments were strted. The seedlings were sujeted then to three wter regimes: field pity (FC), grdul suspension of irrigtion (GS) nd totl suspension of irrigtion (TS). In the FC tretment, the plsti gs were weighed nd the soil mintined ner the field pity. Every three dys, these were weighed one gin for reposition of wter lost y evpotrnspirtion. For the regime of grdul suspension of irrigtion, the wter lost fter the first dy ws restored in 8%, 6%, 4%, 2% nd %, on every three dy intervl. For the regime of totl suspension of irrigtion, the wter ws totlly suspended from the first dy. The experiment ws onduted in ompletely rndomized design with three replites per tretment, nd the evlutions were performed every three dys, during period of 21 dys. The wter sttus ws evluted y mesurements potentil of pre-dwn (efore sunrise) lef wter potentil with Sholnder-type pressure hmer on six fully expnded leves of the fourth pir. Lef trnspirtion (E) nd stomtl resistne (rs) mesurements were tken on six fully expnded leves of the fourth pir with stedy stte porometer t 1 m nd 5 pm. The men vpor pressure defiit (VPD) ws the vpor pressure t lef temperture minus the men vpor pressure of the hmer gs (omputed s the verge vpor pressure of the gs entering nd tht exiting the hmer). The timing of determintions followed the reommendtions of Deuner et l. (28) sine t 1 m the photosynthesis nd trnspirtion were higher nd stomtl resistne ws miniml nd 5 pm there ws the mximum expression of ntioxidnt enzymes. Thus mesurements of stomtl resistne, trnspirtion nd VPD were lso rried out t 5 pm in order to ompre the tivity of enzymes (in its time of mximum expression) with these fetures. At 5 pm, following the reommendtions of (Deuner et l., 28), leves of the third pir were olleted for the iohemil (hydrogen peroxide, lipid peroxidtion, sorte - As nd dehydrosorte DHA) nd enzymti (superoxide dismutse - SOD, tlse - CAT, sorte peroxidse - APX nd glutthione redutse GR) nlyses. The olleted mteril ws frozen in liquid nitrogen nd stored in ultr-freezer, t -8ºC, for susequent nlysis. Hydrogen peroxide (H 2 ) ontent ws determined ording to methodology desried y Sinh et l. Si. Agri. (Piri, Brz.), v.68, n.1, p.77-85, Jnury/Ferury 211

3 Antioxidtive system in offee plnts under wter stress 79 (25). Lef tissues were merted in solution of.1% (w/v) trihloroeti id (TCA) nd the homogente entrifuged t 12, x g for 15 min., t 4ºC. Next,.5 ml of the superntnt ws dded to.5 ml of 1 mm potssium phosphte uffer (ph 7.) nd 1 ml of 1 M KI. Asorne mesurements were rried out in spetrophotometer t 39 nm. The H 2 ontent ws lulted y ompring the reds with stndrd urve otined from onentrtions of H 2. Lipid peroxidtion ws determined y quntifition of the thiorituri id retive speies (TBARS) s desried y Buege nd Aust (1978). Two hundred milligrms of lef tissue were merted in liquid N 2 plus 2% of PVPP (polivinilpolipirolidone) nd homogenized in trihloroeti id (TCA).1% (w/v). The homogente ws then entrifuged t 1, x g for 1 min. Two hundred nd fifty miroliters of the superntnt were dded to 1 ml of the retion medium [.5% (w/v) of thiorituri id (TBA) nd 1% (w/v) of TCA], inuting it then t 95ºC for 3 minutes. The retion ws stopped y rpid ooling in ie, nd the sorne mesurements were determined in spetrophotometer t 535 nm nd 6 nm. The onentrtion of the omplex MDA/TBARS ws lulted using the extintion oeffiient of 5 mmol L 1 m 1. Asorte (As) nd dehydrosorte (DHA) were quntified s desried y Arkw et l. (1981) with some modifitions. One hundred milligrms of lef tissue were merted in 2 ml of trihloroeti id (TCA) 5% (w/v) homogente nd entrifuged t 1, g for 15 min t 4ºC. Totl sorte (As + DHA) ws determined fter redution of DHA y dithiothreitol (DTT). Then, 3 μl of the superntnt were dded to retion medium ontining: 7 μl of 5% TCA (w/v), 125 μl of 6% DTT (w/v) nd 125 μl sodium phosphte.2 M (ph etween 7 nd 8 djusted with 1.2 M NOH). After inution t room temperture for 1 min, 125 μl of N-ethylmleimide.24% (w/ v) ws dded nd the ph of eh tue djusted to etween 1 nd 2 with the ddition of 2% TCA (w/v). After tht, 125 μl of phosphori id (H 3 PO 4 ) 4% (v/v), 25 μl of thophennthroline.5% (w/v) nd 125 μl of FeCl 3 3% (w/v) were dded, mixing the mixture vigorously nd inuting it t 3ºC for 9 minutes. The reds were performed in spetrophotometer t 534 nm. The sorte ws determined s desried ove, ut repling the DTT y solute ethnol in equl volume. The vlues for DHA were otined y the differene etween the vlues of totl sorte nd sorte. Two hundred milligrms of lef tissue were merted in liquid N 2 with 5% of polyvinylpyrrolidone (PVPP) nd homogenized in ml of extrtion uffer s following: 1 mm potssium phosphte (ph 7.8),.1 mm EDTA nd 1 mm sori id. After entrifugtion t 13, g for 1 minutes t 4ºC, the superntnt ws olleted nd deslinted in Sephdex G-25 Column (PD-1). The proteins were eluted with the sme uffer nd monitored t 595 nm. The elute ws used for the enzymes tivity ssys nd quntifition of protein y the method of Brdford (1976). SOD tivity ws ssessed y the ility the enzyme to inhiit the lue of nitrotetrzole (NBT) photoredution (Ginnopolitis nd Ries, 1977) in retion medium omposed of 1 mm potssium phosphte (ph 7.8), 14 mm methionine, EDTA.1 μm, 75 mm NBT nd rioflvin 2 mm. The tues with the retions nd the smples were illuminted for seven minutes in ox y fluoresent lmp of 2 W. For the ontrol, the sme retion medium without the smple ws illuminted nd s lnk ws used the tue kept in the drk. The redings were performed t 56 nm. One unit of SOD is the mount of enzyme ple to inhiit y 5% the photoredution of NBT under the tested onditions. The tivity of CAT ws determined s desried y Azevedo et l. (1998) with minor modifitions. Ativity ws monitored y the derese in sorne t 24 nm for two minutes in retion medium inuted t 28ºC, ontining 1 mm potssium phosphte uffer (ph 7.) nd 1 mm H 2. The tivity of APX ws determined s Nkno nd Asd (1981), trking the oxidtion rte of sorte t 29 nm. The retion medium tht ws inuted t 28ºC ws omposed of 1 mm potssium phosphte uffer (ph 7.), sori id.5 mm nd.1 mm H 2. The derese in sorne ws monitored for period of two minutes from the eginning of the retion. The tivity of GR ws sed on the method of Ckmk et l. (1993), trking the rte of oxidtion of NADPH y the derese in sorne, t 34 nm for two minutes. The retion medium inuted t 28ºC onsisted of 5 mm potssium phosphte uffer (ph 7.8), oxidized glutthione 1 mm nd 75 mm NADPH. Dt sets were sujeted to nlyses of vrine (ANOVA) with three wter regimes nd dys of stress s min ftors. Tukey HSD tests were rried out to determine differenes mong tretment mens, using the STATISTICA softwre (ver. 5., Sttsoft, In. Tuls, OK, USA) Results nd Disussion In plnts t field pity (FC), the wter potentil (Ψ W ) ws onstnt, lose to -.2 MP throughout the whole ssessment period (Figure 1). However, in plnts whih the irrigtion ws suspended grdully (GS), derese ws oserved in Ψ W only t 18 d, when the plnts were no longer reeiving wter for three dys. During this period, the Ψ W dropped to - MP, vlue onsidered ritil for offee plnts (Ren nd Mestri, 2; Oliveir et l., 22; Pinheiro et l., 25; Grisi et l., 28) sine it my use redutions in the photosyntheti rtes (Grisi et l., 28). Following this period, the redution ws entuted rehing vlues lose to - MP t 21 dys. On the other hnd, the totl suspension of irrigtion (TS) promoted rpid derese in wter potentil fter the sixth dy of evlution, rehing -2.4 Si. Agri. (Piri, Brz.), v.68, n.1, p.77-85, Jnury/Ferury 211

4 8 Deuner et l. Ψw (MP) FC GS TS MP t 15 dys nd -3. MP t the end of the experiment. Coffee plnts supports long periods of drought, djusting mehnisms tht led to wter sving, proportionlly to the intensity of wter defiit (Deuner et l., 28; Nsimento et l., 28). The stomtl resistne mesured t 1 m (Figure 2A) nd 5 pm (Figure 2B), in plnts tht were kept t FC, showed no differene during the ssessment period (Figure 2A). However, offee plnts under the regime of GS showed n inrese in stomtl resistne mesured t 1 m strting 12 d fter the onset the experiment, eing this effet more pronouned fter 15 d, when the irrigtion ws ompletely stopped nd when Ψ W rehed -.7 MP (Figure 1). For plnts under TS, differenes from the ontrol FC were oserved fter sixth dy, when Ψ W rehed -.5 MP, nd ontinued to inrese to vlues ner - MP, t 15 th dy, vlue onsidered ritil. Until the end of the experiment, with totl restrition of irrigtion, the stomtl resistne ontinued to inrese, even with the potentil flling elow the ritil vlues. The sensitivity of offee plnts stomt to wter defiit is evidened, sine even smll dereses in Ψ w were suffiient to promote its initil losure (Oliveir et l., 22; Pinheiro et l., 25), whih ontinued until 21 dys, when it rehed -2.3 nd -3. MP for the GS nd TS tretments, respetively. The inrese in stomtl resistne, when the leves were still turgid, shows tht the offee stomt prtiipte tively in the proess of wter loss redution, inititing its losure t the first signs of wter restrition (Pinheiro et l., 25). This proess ertinly enles offee plnts to support long periods of drought, without ompromising its degree of ellulr hydrttion. In generl, vlues of stomtl resistne t 5 pm were higher thn those verified t 1 m (Figure 2B). After nine dys of evlution, there were differenes mong the three wter regimes nd this differene entuted with the progress of the experiment. The reovery in sto Dys Figure 1 Lef wter potentil (Ψw) in the eginning of the morning, in offee seedlings sumitted to three wter regimes: field pity (FC), grdul suspension of irrigtion (GS) nd totl suspension of irrigtion (TS). Vlues re mens ± SE. The letters indite the evlutions where differenes were oserved on the sme dy (Tukey, p < 5). mt opening in the morning (Figure 2A), fter its losured hd ourred in the previous fternoon (Figure 2B), reinfores tht the offee stomt remined funtionl even with the lef under strong dehydrtion (Oliveir et l., 22), suh s tht shown fter 21 d of wter restrition. The trnspirtory rtes virtully did not vry etween the two periods of the dy (Figure 2C nd D) nd for plnts sujeted to wter defiit (GS nd ST) rte inversely proportionl to the stomtl resistne ws oserved (Figure 2A nd B), i.e., while it fell the stomtl resistne inresed. The evportive pttern of leves explins vritions in Ψ W (Figure 1). In the se of ontrol FC plnts, redution in trnspirtion y the 9 th dy ws oserved t 5 pm, s result of the higher evportive demnd during this period (Figure 2E nd F), without using, however, dereses in wter sttus of leves (Figure 1). In generl, the oserved trnspirtion ehvior, t oth 1 m nd 5 pm, refleted the stomtl movement whih, in turn, refleted in the pttern set y the wter potentil (Deuner et l., 28). Pinheiro et l. (25), Deuner et l. (28) nd Nsimento et l. (28) lso onluded tht the losure of stomt ppers to e one of the first strtegies to minimize losses of wter ourring with the trnspirtion under onditions of low lef wter potentils The effets of wter stress on plnts my e medited y the prodution nd umultion of retive oxygen speies, leding to extensive memrnes dmge, nd triggering of lipid peroxidtion proesses (Smirnoff, 1993). In this experiment, the levels of H 2 in leves of plnts sujeted to wter stress were higher thn those oserved in ontrol plnts fter three dys (Figure 3A). In the ourse of the period of stress, the vlues inresed lmost linerly, rehing t the end of the experiment, vlues 33 nd 1% higher thn those of the ontrols, for plnt sumitted to GS nd TS, respetively. The H 2 prodution ours in the plnts tissues under vriety of ftors tht inlude extreme tempertures, exessive exitement energy, wter stress, hevy metl nd pthogen tion (Deuner et l., 28; Gomes- Júnior et l., 26; Pompeu et l., 28). The H 2 prodution my our in plnt ells vi numer of ptwys, suh s NAD(P)H oxidse, peroxidtion of lipids or y the trnsport of eletrons in photosynthesis (Montillet et l., 24). By mens of the nlysis of the ehvior of the H 2 prodution in offee plnts under wter restrition, we found tht its metoli route is tivted efore the plnts show ny signs of the wter defiit, due to the high vlues of Ψ W or due to the low vlues of stomtl resistne or trnspirtion (Figure 2). The umultion of retive oxygen speies, here represented y the levels of H 2, n use peroxidtion of ell memrnes, impiring its funtion nd integrity, with dmge, often irreversile, to the funtioning of the ell. In the urrent se, the lipid peroxidtion (Figure 3B) in ontrol plnts (FC) inresed, until the first six dys, from 8.7 to 11.4 nmol of MDA g 1 MF, remining, Si. Agri. (Piri, Brz.), v.68, n.1, p.77-85, Jnury/Ferury 211

5 Antioxidtive system in offee plnts under wter stress 81 rs (s m 1 ) E (µg m 2 s 1 ) A) Dys C) Dys rs (s m 1 ) E (µg m 2 s 1 ) B) Dys D) Dys VPD (kp) E) Dys VPD (kp) F) Dys Figure 2 Stomtl resistne (r s ), trnspirtory rte (E) nd vpor pressure defiit (VPD) evluted t 1 m (First olumn) nd t 5 pm (Seond olumn), in offee seedlings sumitted to three wter regimes: field pity ( ), grdul suspension of irrigtion ( ) nd totl suspension of irrigtion ( ).Vlues re mens ± SE. The letters indite the evlutions where differenes were oserved on the sme dy (Tukey, p < 5). however, in tht level until the 12 th dy, flling from therefter to its initil levels. Lipid peroxidtion is metoli proess tht ours normlly under nturl onditions (Blokin et l. 23), whih explins the vlues of peroxidtion found in these plnts. Plnts tht were in grdully (GS) nd immeditely (TS) sumitted to irrigtion suspension, showed n inrese in lipid peroxidtion in the first six dys, returning to seline vlues during the 9 th nd the 15 th dy from whih inresed gin until the end of the experiment. During this period the H 2 onentrtion in plnts tht were under grdul (GS) nd totl suspension (TS) of irrigtion, ws 33 nd 1% greter thn tht found in ontrol plnts (FC). Differenes in lipid peroxidtion etween irrigted nd non-irrigted plnts were oserved only fter 15 dys, for plnts with totl suspension of irrigtion (TS) nd 21 dys for plnts in grdul suspension of irrigtion (GS). These inreses ourred when the vlues of wter potentil of plnts with wter defiit fell to - MP (Figure 1), nd when the levels of H 2 in GS nd TS plnts were, respetively, 66 nd 33% higher thn their ontrols (Figure 3A). Therefore, the thresholds rnge of wter potentil tht offee plnts my tolerte from - to - MP. Until this ritil point is rehed, the offee plnts, through stomtl ontrol, mintined its wter sttus t the expense of the preservtion of its memrnes integrity. From therefter, the inrese in Si. Agri. (Piri, Brz.), v.68, n.1, p.77-85, Jnury/Ferury 211

6 82 Deuner et l. H 2 onentrtions with its deleterious effets on the peroxidtion of lipids previls, using ellulr dmge tht, depending on its durtion nd intensity, my ompromise the physiologil proesses. As n exmple, there is greter stomt movement towrds stomtl losure, virtully stopping the trnspirtion (Figure 2), extly when the plnts rehed - MP, i.e. t 15 nd 21 dys for GS nd TS plnts, respetively. In this se, it ws oserved tht plnts under totl suspension irrigtion were, until the end of the experiment, under stress for t lest six dys, while plnts under grdul suspension, rehed the wter stress sttus t fter 21 dys, i.e. t the lst ssessment period. Under norml onditions, plnts usully re well dpted to minimize dmge due to the inevitle formtion of retive oxygen speies in photosynthesis (Grtão et l., 25). However, drought intensifies the formtion of free rdils y limiting the pool of NADP + ville to ept eletrons from photosystem I. Thus, inreses the likelihood of exittion energy trnsfer to the, leding to the prodution of nd 1, whih ret with the memrnes ftty ids, using lipid peroxidtion (Smirnoff, 1993). In some drought tolernt plnts, inrese in the prodution of ntioxidnts my limit lipid peroxidtion (Yoshimur et l., 2; Blokin et l., 23). In this experiment, the levels of sori id nd dehydrosorte, in plnts kept t field pity exhiited little vrition (Figure 3C nd D) during the whole evlution period. However, levels of sori id inresed in plnts sujeted to grdul suspension of irrigtion fter the ninth dy, the highest levels ourring t dys 18 nd 21. For plnts under totl suspension of irrigtion, the vlues were similr to the previous tretment; however, there ws drop t dy 21. The dehydrosorte ontent of plnts in grdul suspension of irrigtion (Figure 3D) lso ws higher thn those found in ontrol plnts fter the ninth dy, remining onstnt until the end of the experiment. The plnts under totl suspension of irrigtion showed inreses in the levels of dehydrosorte in reltion to the ontrol plnts fter the third dy of stress. In these plnts, the highest levels were oserved t 15 nd 18 dys. The protetive effet of ntioxidnts ws suffiient to mintin the funtionlity of the memrnes nd its effets on stomtl movement nd Ψ W for period of 15 nd 18 d for plnts under GS nd TS, respetively. These results onfirm those otined y Deuner et l. (28) who oserved tht pplition of exogenous H 2 nd sori id in offee leves, respetively, redued nd inresed stomtl ondutne. Indution of stomtl losure in vivo hs een ttriuted to the umultion of H 2 in gurd ells in response to ABA (Suhit et l., H 2, μmol g 1 FM A) Dys MDA, nmol g 1 FM B) Dys As, μmol g 1 FM C) Dys DHA, μmol g 1 FM D) Dys Figure 3 Hydrogen peroxide (A), lipid peroxidtion (B), sori id (C) nd dehydrosorte (D) in offee seedlings sumitted to three wter regimes: field pity ( ), grdul suspension of irrigtion ( ) nd totl suspension of irrigtion ( ).Vlues re mens ± SE. Asterisks men differenes (p < 5). Vlues re mens ± SE. The letters indite the evlutions where differenes were oserved on the sme dy (Tukey, p < 5). Si. Agri. (Piri, Brz.), v.68, n.1, p.77-85, Jnury/Ferury 211

7 Antioxidtive system in offee plnts under wter stress 83 24). One umulted in the ells, H 2 tivtes the pssge of lium hnnels in vuole memrne, inresing its onentrtion in the ytosol (Kohler nd Bltt, 22), leding therey to depolriztion of gurd ells, efflux of potssium, loss of turgor nd, onsequently, losing the stomt (Shroeder et l., 21). The tivtion of enzymes of the ntioxidnt system interferes in the levels of some ompounds involved these retions. The sori id, esides eing non-enzymti ntioxidnt, ts s oenzyme of APX nd therefore its interellulr ontent n e hnged in funtion of stress. The sme my our with dehydrosorte, sine it is the produt of the retion of this sme enzyme. Asori id is n essentil omponent of plnt tissues nd hs een the fous of numerous studies regrding the enzymti nd non-enzymti oxidtion in iologil systems. It serves s n exellent ntioxidnt nd plys key role in the H 2 removl y the sorte/glutthione yle nd produes DHA (Arkw et l., 1981). ROS re involved in the oxidtion of sori id to form dehydrosorte, whih is susequently regenerted to sori id gin. Antioxidnts suh s sori id nd glutthione, whih re found in high onentrtions (5-2 mm nd 1-5 mm, respetively) in hloroplsts nd other ellulr omprtments, re importnt for the plnts defense ginst oxidtive stress (Blokhin et l., 23). The tivity of superoxide dismutse (SOD) in plnts under GS differed from ontrol FC fter 12 dys of stress, remining onstnt up to 15 dys, when it strted to inrese until the end of the experiment (Figure 4A). This inrese ourred just when the wter potentil fell from -.7 to - MP, pproximtely (Figure 1) nd when stomtl resistne signifintly inresed for the evlution of 5 pm (Figure 2B). For plnts kept under TS, the tivity of SOD inresed onstntly from the eginning of the experiment until the 15 th dy ompring to the ontrol plnts. After 15 d of stress, this inrese ws higher, oiniding with the vlues of wter potentil onsidered elow the ritil threshold (- MP), nd sudden inreses in stomtl resistne nd lipid peroxidtion. However, in the period etween dys 18 nd 21, there ws derese in SOD tivity whih oinided with the highest vlues of lipid peroxidtion. Among the vrious enzymes involved in the elimintion of ROS, SOD n e onsidered key enzyme nd is usully the first line of defense mehnism ginst oxidtive stress (Grtão et l., 25; Pompeu et l., 28). Wter defiit indued higher tivity of SOD, whih determines the onentrtion of nd H 2, eing entrl in the defense mehnisms required to prevent the formtion of OH rdil (Yoshimur et l., 2). Anlysis y non-denturing PAGE followed y tivity stining, reveled the existene of nine SOD isoenzymes SOD (U mg 1 Prot) APX (mmol ASA min 1 mg 1 Prot) A) Dys C) Dys Si. Agri. (Piri, Brz.), v.68, n.1, p.77-85, Jnury/Ferury 211 CAT (μmol H 2 min 1 mg 1 Prot) B) Dys D) Dys Figure 4 Ativity of the enzymes SOD (A), CAT (B), APX (C) nd GR (D), in offee seedlings sumitted to three wter regimes: field pity ( ), grdul suspension of irrigtion ( ) nd totl suspension of irrigtion ( ).Vlues re mens ± SE. The letters indite the evlutions where differenes were oserved on the sme dy (Tukey, p < 5). GR (μmol NADPH min 1 mg 1 Prot)

8 84 Deuner et l. in offee ells suspension ultures in response to dmium (Gomes-Júnior et l., 26). Therefore, we n not dismiss the possiility tht the inrese in SOD tivity in response to wter stress my hve ourred due to the pperne of other forms of isoenzymes. In this se, not only the vritions in tivity ut lso the isoenzymes my e responsile for the prodution of exess H 2. Although SOD is prt of the first line of djustments of the tolerne to oxidtive stress, its produt, H 2, is lso ROS s hrmful s superoxide. In ny se, higher protetion ginst oxidtive dmge my require rpid metolism of H 2 generted y the tion of SOD. Therefore, for the proper funtioning of the detoxifition of free rdils, susequent ntioxidnt enzymes in the system, suh s tlse (CAT) nd the sorte peroxidse (APX), my work in synhrony to remove H 2 (Grtão et l., 25; Pompeu et l., 28). In this se, CAT (Figure 4B) nd APX (Figure 4C) hd similr ehvior to SOD for plnts under oth stressful onditions. For plnts under totl suspension of irrigtion, higher tivity fter the 15 th dy ws oserved, just when ell dmge ourred, s evidened y lipid peroxidtion. In plnts under grdul suspension, the gretest dmge ws oserved when there ws rupt deline in wter potentil etween 12 nd 21 dys. APX uses sorte s the speifi eletron donor to redue H 2 to wter, generting monodehydrosorte, whih in turn needs to e regenerted gin to sorte to mintin the ntioxidnt system tive. For tht, other enzymti retions re involved, intermedited y glutthione, whih is oxidized (Montillet et l., 24). The mintenne of the pool of redued glutthione for the proess depends on the tivity of glutthione redutse (GR). Here, gin, GR tivity showed similr pttern to tht oserved for other enzymes under study (Figure 4D). The inrese in tivity of the enzymes verified in plnts tht were under grdul or totl suspension of irrigtion is due, proly, to the indution of oxidtive stress used y the wter defiieny ondition. It is known tht for offee plnts tht the wter potentil of - MP is onsidered ritil (Ren nd Mestri, 2; Grisi et l., 28). The pility to mintin, in high levels, the tivity of SOD, CAT nd APX, under onditions of environmentl stress, is essentil for mintining the lne etween the formtion nd the removl of H 2 from the intrellulr environment (Zhng nd Kirkm, 1996). However, redued CAT tivity nd inresed tivity of other peroxidses indite tht in plnts kept under stress onditions, the H 2 generted is preferly onsumed in oxidtive proesses, suh s peroxidtion of lipids, thn eliminted y the metolism (Ckmk et l., 1993). The inresed prodution of ntioxidnts, omined with the tivity of ntioxidnt enzymes, seems to e the min strtegy to limit lipid peroxidtion in plnts (Buege et l., 1978). Thus the tion of these enzymes, in ddition to the tion of low moleulr weight ntioxidnts suh s sorte, my, indeed, eliminte, sweep nd immoilize ROS (Grtão et l., 25). Deuner et l. (28) showed tht in offee spryed with H 2, SOD, CAT, APX nd GR tivities inresed to vlues ove those oserved in ontrol plnts. Furthermore, fter pplition of sori id, SOD remined throughout the evlution period with tivity lose to tht oserved in ontrol plnts. In the finl ssessment the tivity of ll enzymes in plnts under wter defiit delined, proportionlly to the level of stress. In the se of plnts under TS in the penultimte evlution (18 dys), the wter potentil hd exeeded the tolerle vlue of - MP nd higher level of lipid peroxidtion, tht ws entuted in the lst dy, hd lredy een oserved (Figure 3B). For the plnts whih the speed of stress ws delyed (GS), the wter potentil only rehed the ritil vlue t 21 th dy, nd when ompred to ontrol differenes were oserved regrding lipid peroxidtion. Therefore, this derese in tivity of ntioxidtive enzymes my e due to destrution of the ellulr memrne system, proportionlly to the stress level, represented y the low wter potentil nd high onentrtion of hydrogen peroxide, in the penultimte nd finl ssessments of TS nd GS, respetively. Conlusions Eight months old plnts of Ctuí IAC 99 offee, sujeted to the restrition of wter in the soil, triggers mehnisms relted to stomtl losure nd detoxifition of free rdils in order to keep the ellulr hydrtion. Until the 15 th nd 18 th dys of moderte nd severe stress, respetively, there is n inrese in stomtl resistne in response to H 2 elevtion, whih remined t eptle levels, ounterlned y the prodution of ompounds nd synhronized tion of ntioxidnt enzymes. Until then, there ws then n effiient stomtl ontrol, mintining good wter sttus t the expense of the preservtion of the memrnes integrity. With the persistene of stress, there ws n imlne in fvor of prodution nd removl of H 2 until ritil point when the wter potentil fell to - MP ws rehed. Therefter, the priority is the inrese in onentrtions of H 2, with its deleterious effets on the peroxidtion of lipids, leding to stomt losure, prlyzing the trnspirtion. Aknowledgements To FAPEMIG (Fundção de Ampro Pesquis do Estdo de Mins Geris) nd CNPq (Conselho Nionl de Desenvolvimento Científio e Tenológio). Referenes An, Z.; Jing, W.; Liu, Y.; Zhng, W. 28. Hydrogen peroxide generted y opper mine oxidse is involved in sisi idindued stomtl losure in Vii f. Journl of Experimentl Botny 59: Si. Agri. (Piri, Brz.), v.68, n.1, p.77-85, Jnury/Ferury 211

9 Antioxidtive system in offee plnts under wter stress 85 Arkw, N.; Tsutsumi, K.; Sned, N.G.; Kurt, T.; Ingki, C A rpid nd sensitive method for the determintion of sori id using 4,7-diphenyl-1,1-phennthroline. Agriulturl nd Biologil Chemistry 45: Azevedo, R.A.; Als, R.M.; Smith, R.J.; Le, P.J Response from elevted ron dioxide to ir nd ozone fumigtion in leves nd roots of wild type nd tlse-defiient mutnt of rley. Physiologi Plntrum 14: Blokhin, O.; Virolinen, E.; Fgerstedt, K.V. 23. Antioxidnts, oxidtive dmge nd oxygen deprivtion stress: review. Annls of Botny 91: Brdford, M.M A rpid nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein-dye inding. Anlytil Biohemistry 72: Buege, J.A.; Aust, S.D Mirosoml lipid peroxidtion. Methods in Enzymology 52: Ckmk, I.; Str, D.; Mrshner, H Ativities of hydrogen peroxide-svenging enzymes in germintion whet seeds. Journl of Experimentl Botny 44: Deuner, S.; Alves, J.D.; Fries, D.D.; Znndre, I.; Lim, A.A.; Henrique, P.C.; Goulrt, P.F.P. 28. Hydrogen peroxide nd sori id effets on ntioxidnt enzyme tivity in offee seedlings. Revist Ceres 55: (in Portuguese, with strt in English). Freits, R.B.; Alves, J.D.; Mglhães, M.M.; Goulrt, P.F.P.; Nsimento, M.N.; Fries, D.D. 27. Coffee tree fertiliztion with potssium nitrte vi lef nd soil, in utumn-winter nd spring-summer: effets on nitrte redutse tivity, on plnt growth nd prodution. Ciêni e Agrotenologi 31: (in Portuguese, with strt in English). Ginnopolitis, C.N.; Ries, S.K Superoxide dismutses. I. Ourrene in higher plnts. Plnt Physiology 59: Gomes, I.A.C.; Cstro, E.M.; Sores, A.M.; Alves, J.D.; Alvreng, M.I.N.; Alves, E.; Bros, J.P.R.A.D.; Fries, D.D. 28. Morphophysiologil ltertions in leves of Coffe ri L. v. Oeirs shded y Ai mngium Willd. Ciêni Rurl 38: (in Portuguese, with strt in English). Gomes-Júnior, R.A.; Moldes, C.A.; Delite, F.S.; Pompeu, G.B.; Grtão, P.L.; Mzzfer, P.; Le, P.J.; Azevedo, R.A. 26. Antioxidnt metolism of offee ell suspension ultures in response to dmium. Chemosphere 65: Grtão, P.L.; Polle A.; Le P.J.; Azevedo R.A. 25. Mking the life of hevy metl-stressed plnts little esier. Funtionl Plnt Biology 32: Grisi, F.A.; Alves, J.D.; Cstro, E.M.; Oliveir, C.; Bigiotti, G.; Melo, L. 28. Lef ntomil evlutions in Ctuí nd Siriem offee seedlings sumitted to wter stress. Ciêni e Agrotenologi 32: (in Portuguese, with strt in English). Kohler, B.; Bltt, M.R. 22. Protein phosphoryltion tivtes the gurd ell C hnnel nd is requisite for gting y sisi id. Plnt Journl 32: Montillet, J.L.; Cs, J.L.; Grnier, L.; Montne, M.H.; Douki, T. 24. The upstrem oxylipin prowle of Aridopsis thlin: tool to sn for oxidtive stresses. Plnt Journl 4: Nsimento, M.N.; Alves, J.D.; Sores, A.M.; Cstro, E.M.; Mglhães, M.M.; Alvreng, A.A.; Silv, G.H. 28. Biohemil ltertions of plnts nd ud morphology of offee tree ssoited to events on flowering in response to meteorologil elements. Ciêni Rurl 38: (in Portuguese, with strt in English). Oliveir, M.A.J.de; Bovi, M.L.A.; Mhdo, E.C.; Gomes, M.M. de A.; Hermnn, G.; Rodrigues, J.D. 22. Photosynthesis, stomtl ondutne nd trnspirtion in peh plm under wter stress. Sienti Agriol 59: (in Portuguese, with strt in English). Pereir, S.P.; Guimrães, R.J.; Brtholo, G.F.; Guimrães, P.T.G.; Alves, J.D. 27. Vegettive growth nd yield of offee plnts (Coffe ri L.) in two different pruning times, onduted t different spings. Ciêni e Agrotenologi 31: (in Portuguese, with strt in English). Pinheiro, H.A.; DMtt, F.M.; Chves, A.R.M.; Loureiro, M.E.; Dutti, C. 25. Drought tolerne is ssoited with rooting depth nd stomtl ontrol of wter use in lones of Coffe nephor. Annls of Botny 96: Pompeu, G.B.; Grtão, P.L.; Vitorello, V.A.; Azevedo, R.A. 28. Antioxidnt isoenzyme responses to nikel-indued stress in too ell suspension ulture. Sienti Agriol 65: Ren, A.B.; Mestri, M. 2. Wter reltions in offee. ITEM 48: (in Portuguese). Sinh, S.; Sxen, R.; Singh, S. 25. Chromium indued lipid peroxidtion the plnts of Pisti strtiotes L.: Role of ntioxidnts nd ntioxidnt enzymes. Chemosphere 58: Smirnoff, N The role of tive oxygen in the response of plnts to wter defiit nd desition. New Phytologist 125: Shroeder, J.I.; Kwk, J.M.; Allen, G.J. 21. Gurd ell sisi id signling nd engineering drought hrdiness in plnts. Nture 41: Suhit, D.; Rghvendr, A.S.; Kwk, J.M.; Vvsseur, A. 24. Cytoplsmi lkliztion preedes ROS prodution during methyl jsmonte- nd sisi id-indued stomtl losure. Plnt Physiology 134: Vsonelos, A.C.F.; Zhng, X.; Ervin, E.H.; Kiehl, J.C. 29. Enzymti ntioxidnt responses to iostimulnts in mize nd soyen sujeted to drought. Sienti Agriol 66: Zhng, J.; Kirkm, M.B Lipid peroxidtion in sorghum nd sunflower seedlings s ffeted y sori id, enzoi id nd propyl gllte. Journl of Plnt Physiology 149: Zhng, X.; Ervin, E.; Evnylo, G.; Sherony, C.; Peot, C. 25. Biosolids impt on tll fesue drought resistne. Journl of Residuls Siene & Tehnology 2: Zhng, X.; Zhng, L.; Dong, F.; Go, J.; Glrith, D.W.; Song, C.P. 21. Hydrogen peroxide is involved in sisi id-indued stomtl losure in Vii f. Plnt Physiology 126: Reeived July 14, 29 Aepted My 19, 21 Si. Agri. (Piri, Brz.), v.68, n.1, p.77-85, Jnury/Ferury 211