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Journal of Plant Physiology 162 (2005) 1114—1122

KEYWORDAcclimatioAntioxidatzymes;HydrogenLipid peroMaize;OxidativeSalt stressZea mays

0176-1617/$ - sdoi:10.1016/j.

Abbreviationglutathione; GSchloride; RDM,�CorrespondE-mail addr

www.elsevier.de/jplph

Hydrogen peroxide pre-treatment induces salt-stress acclimation in maize plants

Andre Dias de Azevedo Netoa, Jose Tarquinio Priscob, Joaquim Eneas-Filhob, Jand-Venes Rolim Medeirosb, Eneas Gomes-Filhob,�

aDepartamento de Biologia, Universidade Federal Rural de Pernambuco, 52171-900, Recife, Pernambuco, BrazilbDepartamento de Bioquımica e Biologia Molecular, Universidade Federal do Ceara, Caixa Postal 6039, 60455-900Fortaleza, Ceara, Brazil

Received 28 October 2004; accepted 5 January 2005

Sn;ive en-

peroxide;xidation;

stress;;

ee front matter & 200jplph.2005.01.007

s: APX, ascorbate peSG, oxidized glutathiroot dry mass; ROS, ring author. Tel.: +55 8ess: egomesf@ufc.br

SummaryThe effect of exogenously applied H2O2 on salt stress acclimation was studied withregard to plant growth, lipid peroxidation, and activity of antioxidative enzymes inleaves and roots of a salt-sensitive maize genotype. Pre-treatment by addition of 1mMH2O2 to the hydroponic solution for 2 days induced an increase in salt tolerance duringsubsequent exposure to salt stress. This was evidenced by plant growth, lipidperoxidation and antioxidative enzymes measurements. In both leaves and roots thevariations in lipid peroxidation and antioxidative enzymes (superoxide dismutase,ascorbate peroxidase, guaiacol peroxidase, glutathione reductase, and catalase)activities of both acclimated and unacclimated plants, suggest that differences in theantioxidative enzyme activities may, at least in part, explain the increased toleranceof acclimated plants to salt stress, and that H2O2 metabolism is involved as signal inthe processes of maize salt acclimation.& 2005 Elsevier GmbH. All rights reserved.

Introduction

Cellular homeostasis is achieved by the coordi-nated action of many biochemical pathways.However, under suboptimal conditions stress

5 Elsevier GmbH. All rights rese

roxidase; CAT, catalase; GPX,one; H2O2, Hydrogen peroxide; Leactive oxygen species; SDM, sh5 3288 9405; fax: +55 85 3288 982(E. Gomes-Filho).

different pathways can be affected, and theircoupling, which makes cellular homeostasis possi-ble, is disrupted (Rizhsky et al., 2002). This processis usually accompanied by the formation of reactiveoxygen species (ROS) resulting from an increased

rved.

guaiacol peroxidase; GR, glutathione reductase; GSH, reducedA, leaf area; MDA, malondialdehyde; NBT, nitro blue tetrazoliumoot dry mass; SOD, superoxide dismutase9.

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H2O2 pre-treatment in salt-stressed maize 1115

flow of electrons from the disrupted pathways tothe reduction of oxygen (Halliwel and Gutteridge,1989; Noctor and Foyer, 1998; Asada, 1999; Dat etal., 2000; Mittler, 2002). ROS formation leads tochanges in intracellular redox homeostasis (Bowlerand Fluhr, 2000), and it is now widely accepted thatredox signals are key regulators of plant metabo-lism, morphology and development (Foyer andNoctor, 2003).

The ROS hydrogen peroxide (H2O2) has generallybeen viewed as a toxic cellular metabolite. How-ever, it is now clear that it may also function as asignal molecule in both plant and animal cells(Finkel, 2000; Neill et al., 2002b). The generationof H2O2 is increased in response to a wide variety ofabiotic and biotic stresses, and some authors havesuggested that H2O2 plays a dual role in plants: atlow concentrations, it acts as a messenger mole-cule involved in acclimatory signaling, triggeringtolerance against various abiotic stresses, and athigh concentrations it orchestrates programmedcell death (Prasad et al., 1994; Van Breusegem etal., 2001; Neill et al., 2002a; Vandenabeele et al.,2003). Thus, it appears likely that H2O2 accumula-tion in specific tissues, and in the appropriatequantities, may benefit plants by mediating plantacclimation and cross-tolerance to both biotic andabiotic stresses (Bowler and Fluhr, 2000).

Several studies have produced evidence for asignaling role for H2O2 (Foyer et al., 1997). Theaddition of H2O2 to plant tissues or its experimentalgeneration has been demonstrated to act as asignal in the induction of gene expression of theenzymes catalase (CAT) (Prasad et al., 1994;Polidoros and Scandalios, 1999), ascorbate perox-idase (APX) (Van Breusegem et al., 2001), guaiacolperoxidase (GPX) and glutathione reductase (GR)(Janda et al., 1999). Changes in H2O2 homeostasisalso induces synthesis of heat shock proteins andactivates the mitogen-activated protein kinasecascade (Kovtun et al., 2000; Van Breusegem etal., 2001). In addition, Desikan et al. (2001) usingcDNA microarray technology identified 175 non-redundant expressed sequence tags that are regu-lated by H2O2.

Endogenous H2O2 production has been shown toincrease in response to chilling stress in maizeseedlings, and exogenously applied H2O2 increasedchilling stress tolerance. This increase in tolerancewas partly due to an enhanced antioxidative systemthat prevents the accumulation of ROS duringchilling stress (Prasad et al., 1994). Nodal potatoexplants subcultured from H2O2-treated micro-plants (acclimated) were resistant to a 15-h heatshock at 42 1C (a normally lethal treatment) evenafter 4 weeks of treatment (Lopez-Delgado et al.,

1998). H2O2 injection in Arabidopsis leaves led toprotection from a subsequent excess light-inducedphotobleaching effect (Karpinski et al., 1999).Recently, important evidence that H2O2 can induceacclimation to salt and heat stresses was obtainedwith rice seedlings pre-treated with various levelsof H2O2 (Uchida et al., 2002). However, it is notknown if H2O2 pre-treatment also induces saltstress tolerance in maize plants.

Considering that changes in H2O2 homeostasismay be a pivotal signaling event of the acclimationphenomenon and a key component in the stresssurvival process, the aim of this study was toevaluate the effects of H2O2 pre-treatment onplant growth, lipid peroxidation and activity ofantioxidative enzymes in leaves and roots of a salt-sensitive maize genotype, in order to betterunderstand the physiological and biochemicalmechanisms of salt acclimation in maize plants.

Materials and methods

Plant material, growth and treatmentconditions

Seeds of BR-5011, a salt-sensitive maize (Zeamays L.) genotype (Azevedo Neto et al., 2004),were sown in trays containing vermiculite andirrigated daily with distilled water. Four days fromseedling emergence they were transferred to trayscontaining aerated half-strength Hoagland’s nutri-ent solution. Three days later half of the plantswere pre-treated with nutrient solution containing1.0 mM H2O2 for 2 days (acclimation treatment)while the other half remained in nutrient solution.Preliminary studies using various concentrations ofH2O2 (from 0.1 to 1000 mM H2O2) and two differenttimes of pre-treatment (1 or 2 days) have shownthat 1.0 mM H2O2 for 2 days was closer to theoptimum combination to acclimate the seedlings.The plants were then transferred to 3 L plastic potscontaining full-strength nutrient solution with orwithout 100mM NaCl. Therefore, the plants weresubmitted to four treatments: control (not pre-treated with H2O2 and not salt-stressed); unaccli-mated-stressed (not pre-treated with H2O2 andsalt-stressed); acclimated-unstressed (pre-treatedwith H2O2 and not salt-stressed); and acclimated-stressed (pre-treated with H2O2 and salt-stressed).Addition of salt (25mM per day) began on trans-planting into plastic pots. The experiment wascarried out under glasshouse conditions. The meanvalues of temperature, relative air humidity andphotosynthetic active radiation (at noon) were

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A.D. de Azevedo Neto et al.1116

27 1C, 65% and 1200 mmolm�2 s�1, respectively.Plants were harvested just before pre-treatment(day 0), at the end of pre-treatment (before thestart of salt additions – day 2), and at 1 (day 6), 5(day 10) and 10 (day 15) days after the end of saltadditions. Plants from each treatment were sepa-rated into shoot and roots for dry mass (DM)determinations as well as leaf area measurementsas described in Azevedo Neto et al. (2004). The firstfully expanded leaf and the younger third of theroot system were frozen in liquid nitrogen, lyophi-lized, ground to a powder and kept in a freezer(�25 1C) for further biochemical analyses.

Extract preparation

Lyophilized leaf (0.20 g) and root (0.15 g) powderwere homogenized in a mortar and pestle with 4mLof ice-cold extraction buffer (100mM potassiumphosphate buffer, pH 7.0, 0.1mM EDTA). Thehomogenate was filtered through muslin cloth andcentrifuged at 16,000g for 15min. The supernatantfraction was used as crude extract for enzymeactivity and lipid peroxidation assays. All opera-tions were carried out at 4 1C.

Enzyme assays

Total superoxide dismutase (SOD) (EC 1.15.1.1)activity was determined by measuring its ability toinhibit the photochemical reduction of nitro bluetetrazolium chloride (NBT), as described by Gian-nopolitis and Ries (1977). The reaction mixture(1.5mL) contained 50mM phosphate buffer (pH7.8), 0.1 mM EDTA, 13mM methionine, 75 mM NBT,2 mM riboflavin and 50 mL enzyme extract. Ribo-flavin was added last and tubes were shaken andilluminated with a two 20-W fluorescent tubes. Thereaction was allowed to proceed for 15min afterwhich the lights were switched off and the tubescovered with a black cloth. Absorbance of thereaction mixture was read at 560 nm. One unit ofSOD activity (U) was defined as the amount ofenzyme required to cause 50% inhibition of the NBTphotoreduction rate and the results expressed asUmg�1 of dry mass (DM).

Total CAT (EC 1.11.1.6) activity was measuredaccording the method of Beers and Sizer (1952),with minor modifications. The reaction mixture(1.5mL) consisted of 100mM phosphate buffer (pH7.0), 0.1 mM EDTA (20mM H2O2 and 50 mL enzymeextract. The reaction was started by addition of theextract. The decrease of H2O2 was monitored at240 nm and quantified by its molar extinction

coefficient (36M�1 cm�1) and the results expressedas mmol H2O2 min�1 g�1 DM.

Total APX (EC 1.11.1.1) activity was assayedaccording to Nakano and Asada (1981). The reac-tion mixture (1.5mL) contained 50mM phosphatebuffer (pH 6.0), 0.1 mM EDTA, 0.5mM ascorbate,1.0mM H2O2 and 50 mL enzyme extract. Thereaction was started by addition of H2O2 andascorbate oxidation measured at 290 nm for1min. Enzyme activity was quantified using themolar extinction coefficient for ascorbate(2.8mM�1 cm�1) and the results expressed in mmolH2O2 min�1 g�1 DM, taking into consideration that2mol ascorbate are required for reduction of 1molH2O2 (McKersie and Leshem, 1994).

Total GPX (EC 1.11.1.7) activity was determinedas described by Urbanek et al. (1991) in a reactionmixture (2.0mL) containing 100mM phosphatebuffer (pH 7.0), 0.1 mM EDTA, 5.0mM guaiacol,15.0mM H2O2 and 50 mL enzyme extract. Thereaction was started by addition of enzyme extractand the increase in absorbance recorded at 470 nmfor 1min. Enzyme activity was quantified by theamount of tetraguaiacol formed using its molarextinction coefficient (26.6mM�1 cm�1). The re-sults were expressed as mmol H2O2 min�1 g�1 DMtaking into consideration that 4mol H2O2 arereduced to produce one mol tetraguaiacol (Plewaet al., 1991).

Total GR activity (EC 1.6.4.2) was assayed asdescribed by Foyer and Halliwell (1976), with minormodifications. The reaction mixture (1.0mL) con-sisted of 100mM phosphate buffer (pH 7.8), 0.1 mMEDTA, 0.05mM NADPH, 3.0mM oxidized glutathione(GSSG) and 50 mL enzyme extract. The reaction wasstarted by the addition of GSSG and the NADPHoxidation rate was monitored at 340 nm for 1.0min.Enzyme activity was determined using the molarextinction coefficient for NADPH (6.2mM�1 cm�1)and expressed as mmol NADPH min�1mg�1 DM.

Lipid peroxidation

Lipid peroxidation was determined by measuringthe amount of malondialdehyde (MDA) produced bythe thiobarbituric acid reaction as described byHeath and Packer (1968). The crude extract wasmixed with the same volume of a 0.5% (w/v)thiobarbituric acid solution containing 20% (w/v)tricholoroacetic acid. The mixture was heated at95 1C for 30min and then quickly cooled in an ice-bath. The mixture was centrifuged at 3000g for10min and the absorbance of the supernatant wasmonitored at 532 and 600 nm. After subtractingthe non-specific absorbance (600 nm), the MDA

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g pl

-1

3.2

4.8

6.4

SDM

H2O2 pre-treatment in salt-stressed maize 1117

concentration was determined by its molar extinc-tion coefficient (155mM�1 cm�1) and the resultsexpressed as mmol MDA g�1 DM.

The experimental design was a completelyrandomized factorial, five (harvest times)� two(H2O2 levels)� two (salt levels) with five repli-cates. In the biochemical analyses, for each extractthe absorbance was determined on duplicateassays. The data were expressed as the mean-s7standard deviation.

0.0

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g pl

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1.6

Time (days)

0 2 6 10 15

m2

pl-1

0.00

0.04

0.08

0.12

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LA

RDM

Fig. 1. Time course of changes in shoot dry mass (SDM),root dry mass (RDM), and leaf area (LA) of maize plants.The plants were submitted to four treatments: control(J); unacclimated-stressed (�); acclimated-unstressed(,); and acclimated-stressed (.). Plants were harvestedbefore pre-treatment (day 0), at the end of pre-treatment and just before the start of the salt additions(day 2), and 1 (day 6), 5 (day 10) and 10 (day 15) daysafter the end of salt additions. Values indicate themean7SD.

Results

Plant growth

The time course of changes in shoot dry mass(SDM), root dry mass (RDM), and leaf area (LA) aregiven in Fig. 1. It may be observed that unaccli-mated-stressed plants had their SDM, RDM and LAreduced when compared to the control treatment.These reductions were 52%, 32% and 50% at day 10,and 45%, 30% and 48% at day 15, respectively.However, the salt-induced growth inhibition de-creased when the seedlings were pre-treated withH2O2. When comparing acclimated-stressed withunacclimated-stressed plants it can be seen thatthere were increases in SDM, RDM and LA of 35%,26% and 23% at day 10, and 22%, 15% and 24% at day15, respectively. The data also show that the H2O2

pre-treatment by itself did not affect the plantgrowth in relation to control treatment.

Enzyme activity

The time course of total SOD activity in leavesshowed a transient increase at the end of acclima-tion (day 2), and after day 6 it remained unchangeduntil the end of the experimental period (Fig. 2A).SOD activity in both acclimated-unstressed andacclimated-stressed plants was, respectively, 27%and 42% higher than in control or unacclimated-stressed plants. In roots, SOD activity in acclimatedplants was 21% higher than in control plants at 2days from the start of acclimation, reached amaximum at day 6 and then decreased up to day 10in all treatments (Fig. 2B). Salt stress increasedroot SOD activity in both acclimated and unaccli-mated plants at the time of maximal activity (day6), but this salt-induced effect appeared to bereverted at day 10. From day 10 up to the end ofthe experimental period the variations in enzymeactivity show that neither salt stress nor the H2O2

pre-treatment had any significant effect on rootSOD activity.

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Time (days)

U m

g-1

DM

00 02 2 6 10 156 10 15

2

4

6

8

Time (days)

A B

Fig. 2. Time course of total SOD activity in leaves (A) and roots (B) of maize plants. Additional details as in Fig. 1.

µmol

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GRGPX

10 15 10 15

Fig. 3. Time course of total CAT, APX, GPX, and GR activities in leaves of maize plants. Additional details as in Fig. 1.

A.D. de Azevedo Neto et al.1118

The time courses of total CAT, APX, GPX, and GRactivities in leaves are shown in Fig. 3. CAT activityincreased by 73% as a result of H2O2 pre-treatmentat day 2. The enzyme activity in control plantsshowed a slight variation up to day 10, and thenthere was an increase up to the end of theexperimental period. The unacclimated-stressedplants showed the same general pattern of thecontrol plants. CAT activity in acclimated-un-stressed plants did not differ of control andunacclimated-stressed plants from day 10 up tothe end of the experimental period. During thesame period the activity of the acclimated-stressedplants was approximately 42% (day 10) and 60% (day15) higher than in the other treatments. APXactivity remained fairly constant throughout theexperimental period in both control and in accli-

mated-unstressed plants. However, salt stressinduced a marked increase in the APX activity inboth acclimated and unacclimated plants. The salt-induced increases in APX activity were nearly 150%at day 10 and 290% at day 15, when compared toboth control and acclimated-unstressed plants. Saltstress also induced increase in GPX activity, eitherin acclimated or unacclimated plants, however thiseffect was more conspicuous in acclimated ones.GR activity slightly decreased in all treatments upto day 6 from the start of the H2O2 pre-treatment.During the latter part of the experimental periodGR followed the same pattern as APX activity. Theenzyme activities in the salt stress treatments wereabout 35% higher than those of the control and ofthe acclimated-unstressed plants, at the end of theexperimental period.

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µmol

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Fig. 4. Time course of total CAT, APX, GPX, and GR activities in roots of maize plants. Additional details as in Fig. 1.

H2O2 pre-treatment in salt-stressed maize 1119

The time course of total CAT, APX, GPX, and GRactivities in roots are shown in Fig. 4. CAT, APX,GPX, and GR activities in the acclimated plantswere 70%, 26%, 22% and 17% higher than in controlplants, respectively, at 2 days after the start ofacclimation. CAT and GPX activities followed thesame general trend throughout the experimentalperiod in all treatments, i.e., they increased up today 6, remained fairly constant at day 10, andincreased at day 15. There was only one exception,i.e., while the enzyme activities in all treatmentsincreased from the 10th to the 15th day,CAT activity in the unacclimated-stressedplants remained constant. Because of this,at the end of the experimental period, CAT activityin roots of acclimated-stressed plants was 170%higher than in unacclimated-stressed ones. Thetime course of APX and GR activities in rootsfollowed similar trends, and substantial differenceswere not observed among the treatments afterday 6.

Comparing activities of H2O2-scavenging enzymesin leaves (Fig. 3) of salt-stressed plants at the endof the experimental period, CAT activity was 4.6-and 38.5-fold greater than GPX and APX activities,respectively. On the other hand, in roots, GPXactivity (Fig. 4) in salt-stressed plants was 7.5- and70-fold greater than CAT and APX activities,respectively. It is noteworthy that GPX activitywas much higher than in roots than in leaves forboth control and salt-stressed plants.

Lipid peroxidation

The time course of lipid peroxidation of bothleaves and roots, measured as MDA content, isshown in Fig. 5. MDA content changed during theexperimental period in leaves of control andacclimated-unstressed plants. In stressed treat-ments, the MDA content increased by nearly 32%at 10 days and 74% at 15 days, when compared tocontrol and acclimated-unstressed plants, respec-tively (Fig. 5A). In roots, the amount of MDA inacclimated plants increased (about 34%) relative tocontrol plants, at 2 days from the beginning of theH2O2 pre-treatment, and then decreased (Fig. 5B).

Discussion

Plant growth (SDM, RDM, and LA) was inhibitedby salinity (Fig. 1). However, it was shown that H2O2

pre-treatment decreased the deleterious effects ofsalt stress on the growth of maize. The data suggestthat H2O2 added to the growth solution for 2 daysbefore the salt additions, led to a salt stressacclimation process. Although acclimation is con-sidered a complex phenomenon, our results indi-cated that while salt stress could be detrimental tounacclimated plants, the previous application of amild oxidative stress could be responsible forreducing the deleterious effects of salinity on plantgrowth.

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Time (days)0 6 10 15

µmol

MD

A g

-1 D

M

0.00

0.05

0.10

0.15

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Time (days)0 6 10 152 2

A B

Fig. 5. Time course of changes in total malondialdehyde content in leaves (A) and roots (B) of maize plants. Additionaldetails as in Fig. 1.

A.D. de Azevedo Neto et al.1120

Superoxide radicals (O2� �) are toxic byproducts of

oxidative metabolism and can interact with H2O2 toform highly reactive hydroxyl radicals (OH � ), whichare thought to be primarily responsible for oxygentoxicity in the cell (Bowler et al., 1992). Thedismutation of O2

� � into H2O2 and oxygen is animportant step in protecting the cell, and iscatalyzed by SOD. We have shown that SOD activityincreased in leaves of both acclimated-stressed andacclimated-unstressed plants (Fig. 2), suggestingthat H2O2 pre-treated plants had a better O2

� �

radical scavenging ability. It has been shown thatsalt tolerance is directly related to the increase inSOD activity (Gosset et al., 1994; Hernandez et al.,2000; Shalata et al., 2001). In addition, overexpression of Mn-SOD in chloroplasts of rice (Tanakaet al., 1999) and Cu/Zn-SOD in chloroplasts oftobacco plants (Badawi et al., 2004) producedenhanced tolerance to salt stress.

SOD initiates detoxification of O2� � by forming

H2O2, which also being toxic must be eliminated byconversion to H2O in subsequent reactions. Inplants, a number of enzymes regulate H2O2

intracellular levels, but CAT, APX, and GPX areconsidered the most important (McKersie andLeshem, 1994; Noctor and Foyer, 1998). GR alsoplays a key role in oxidative stress by convertingthe GSSG into reduced glutathione (GSH). Theelevated levels of GR activity could increase theGSH/GSSG ratio, which is required for ascorbateregeneration and activation of several CO2 fixingenzymes in the chloroplasts (Crawford et al.,2000).

Comparing control and acclimated unstressedplants it may be seen that acclimation by itself didnot induce CAT, APX, GPX, and GR activities inleaves, except for a transient increase in CATactivity at day 2 (Fig. 3). In addition, high leaf APXand leaf GR activities appeared to be induced by

salt stress, while high leaf CAT activity appeared toresult from H2O2-induced acclimation. On the otherhand, enhancement of leaf GPX activity appearedto be correlated with both salt stress and H2O2-induced acclimation.

In roots, the activities of all antioxidativeenzymes were stimulated by H2O2 pre-treatmentat day 2 (Fig. 4). In addition, root CAT activity atday 15 was reduced in unacclimated-stressed andincreased in acclimated-stressed, when comparedto control plants. Our data suggest that H2O2 pre-treatment could directly or indirectly activateantioxidative enzymes during salt stress, leadingto a higher ability to withstand salt-inducedeffects. The results also suggest a general adaptivedefense response of the root system to H2O2-imposed oxidative stress (Liang et al., 2003),considering that the root was the first organdirectly exposed to hydrogen peroxide.

It is noteworthy that the enhancement in leafSOD activity of acclimated-stressed plants (Fig. 2A)was accompanied by an increase in leaf CAT, APX,GPX, and GR activities (Fig. 3), indicating that instressed plants the H2O2 scavenging mechanism wasmore effective in acclimated than in unacclimatedplants. Thus, the results suggests that CAT, APX andGPX activities coordinated with SOD activity mayplay a central protective role in the O2

� � and H2O2

scavenging process (Mittova et al., 2002; Liang etal., 2003; Badawi et al., 2004) and the activeinvolvement of these enzymes is related, at least inpart, to salt-induced oxidative stress tolerance inmaize plants.

In the present study CAT and GPX were, respec-tively, the H2O2 scavenging enzymes with greatercatalytic activity in leaves and roots irrespective ofthe treatment. Therefore, it could be hypothesizedthat CAT is the most important among theH2O2 scavenging enzymes in leaves, while the

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H2O2 pre-treatment in salt-stressed maize 1121

non-specific peroxidase (GPX) seems to play a keyrole in roots. The observation that CATwas the mostupregulated enzyme in both leaves (73%) and roots(70%) at the end of acclimation (day 2) is anadditional support to above conclusion. It alsoindicates that CATappears to be the most importantantioxidative enzyme for overcoming acclimation-imposed oxidative stress (Prasad et al., 1994).

Salt stress is known to result in extensive lipidperoxidation, which has often been used asindicator of salt-induced oxidative membranedamages (Hernandez and Almansa, 2002). Ourresults showed that MDA content increased insalt-stressed leaves of both acclimated and unac-climated plants, but was practically unaffected inroots (Fig. 5). Although some results correlatinglipid peroxidation to antioxidative system activityhave been reported (Hernandez and Almansa,2002; Liang et al., 2003), the observation that lipidperoxidation in leaves of acclimated-stressedplants was not reduced by acclimation-inducedupregulation of the antioxidative system is contraryto this hypothesis. In acclimated roots, the increaseof MDA content at day 2, in spite of the increase inall antioxidative enzyme activities, further sup-ports the above results. Our data also show thatplant growth was not correlated to lipid peroxida-tion under salt stress conditions, since the growthof acclimated-stressed plants was greater than inunacclimated-stressed plants.

Our results show that H2O2 pre-treatment undernormal growth conditions enhanced salt tolerancein the salt-sensitive maize genotype, and thatdifferences in the antioxidative enzyme activitiesmay, at least in part, explain the increasedtolerance of acclimated plants to salt stress.Additional evidence is provided here that H2O2

metabolism is involved as signal in the processes ofmaize salt acclimation. Other studies have shownevidence for a signaling role of H2O2, i.e. theaddition of H2O2 to maize seedlings before coldtreatment increased their chilling tolerance (Pra-sad et al., 1994), and provided some protectionagainst both heat and salt stress conditions in rice(Uchida et al., 2002). Similarly, exogenous H2O2

treatments simultaneously enhanced multi-resis-tance to heat, chilling, drought and salt stresses inmaize seedlings (Gong et al., 2001). Because manystress conditions cause cellular redox imbalances ithas been proposed that oxidative stress defenseresponses might be a central component mediatingcross-tolerance (Bowler et al., 1992), that is,previous exposure to one stress can induce toler-ance to the same or to a different kind of stressexposure (Bowler and Fluhr, 2000). Thus, plantscould make use of common pathways and compo-

nents in the stress–response relationship (Pastoriand Foyer, 2002).

Acknowledgements

We thank Dr. Ladaslav Sodek for reviewing theEnglish text, and acknowledge the financial supportgiven by Conselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq) and Coordenac-aode Aperfeic-oamento de Pessoal de Nıvel Superior(CAPES).

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