Po

OD

a

ARRA

KCDAD

1

rmpHkpgawos

srNmGf

0d

Environmental and Experimental Botany 66 (2009) 487–492

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

hysiochemical and antioxidant responses of the perennial xerophyte Capparisvata Desf. to drought

zden Ozkur, Filiz Ozdemir, Melike Bor, Ismail Turkan ∗

epartment of Biology, Science Faculty, Ege University, Bornova 35100, Izmir, Turkey

r t i c l e i n f o

rticle history:eceived 7 November 2008eceived in revised form 31 March 2009ccepted 19 April 2009

eywords:aperrought stressntioxidative enzymesrought tolerance

a b s t r a c t

Caper (Capparis ovata Desf.) is a perennial shrub (xerophyte) and drought resistant plant which is welladapted to Mediterranean Ecosystem. In the present study we investigated the plant growth, relativewater content (RWC), chlorophyll fluorescence (FV/FM), lipid peroxidation (TBA-reactive substances con-tent) as parameters indicative of oxidative stress and antioxidant enzymes such as superoxide dismutase(SOD), ascorbate peroxidase (APX), peroxidase (POX), catalase (CAT) and glutathione reductase (GR) inrelation to the tolerance to polyethylene glycol mediated drought stress in C. ovata seedlings. For inductionof drought stress, the 35 days seedlings were subjected to PEG 6000 of osmotic potential −0.81 MPa for14 days. Lipid peroxidation increased in PEG stressed seedlings as compared to non-stressed seedlingsof C. ovata during the experimental period. With regard to vegetative growth, PEG treatment caused

decrease in shoot fresh and dry weights, RWC and FV/FM but decline was more prominent on day 14 ofPEG treatment. Total activity of antioxidative enzymes SOD, APX, POX, CAT and GR were investigated in C.ovata seedlings under PEG mediated drought. Induced activities of SOD, CAT and POX enzymes were highand the rate of increment was higher in stressed seedling. APX activity increased on both days of PEGtreatment, however, increase in GR activity was highest on day 14 of drought stress. We concluded thatincreased drought tolerance of C. ovata is correlated with diminishing oxidative injury by functioning of

her r

antioxidant system at hig

. Introduction

Drought, in conjunction with coincident high temperature andadiation, is considered as one of the most important environ-ental extremes that constraints to plant survival and to crop

roductivity in arid- and semi-arid regions (Chaves et al., 2003).owever, to cope with such combination of stresses, which arenown as drought stress, some plants such as Mediterranean xero-hytes exhibit distinct resistant mechanisms which make themood systems to understand physiological and biochemical mech-

nisms underlying drought tolerance of plants. These mechanismshich are based upon osmotic adjustment, regulation of stomatal

pening, modification of cell wall characteristics and extensive rootystem (Rhizopoulou and Psaras, 2003), all, involve in drought tol-

Abbreviations: RWC, relative water content; ROS, reactive oxygen species; SOD,uperoxide dismutase; CAT, catalase; APX, ascorbate peroxidase; GR, glutathioneeductase; POX, peroxidase; PEG, polyethylene glycol; BSA, bovine serum albumin;BT, nitroblue tetrazolium; EDTA, ethylenediamine-N,N,N′ ,N′-tetraacetic acid; MDA,alondialdehyde; DAB, diamino-benzidine tetra-hydrochloride; GSH, glutathione;SSG, oxidized glutathione; GDH, glutamate dehydrogenase; DW, dry weight; FW,

resh weight; PAGE, polyacrylamide gel electrophoresis.∗ Corresponding author. Tel.: +90 232 3884000x2443; fax: +90 232 3881036.

E-mail address: ismail.turkan@ege.edu.tr (I. Turkan).

098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2009.04.003

ates under drought stress.© 2009 Elsevier B.V. All rights reserved.

erance of these plants, particularly during the summer when lowwater availability is superimposed on high light and high temper-atures at mid-day (Munne-Bosch and Penuelas, 2004).

Such as other environmental stressors, drought stress causesalso oxidative stress due to decreased stomatal conductivity whichrestricts CO2 influx in to the leaves. Decreased leaf internal CO2leads to the formation of reactive oxygen species (ROS) such as rad-ical (O2

•−), hydroxyl radical (OH), hydrogen peroxide (H2O2) andalkoxyl radical (RO) by enhanced leakage of electrons to molec-ular oxygen. Chloroplasts, mitochondria and peroxisomes are themajor source of ROS in plant cells (Asada, 1999). Reactive oxygenspecies have long been proposed as signal molecules that reg-ulate various processes such as growth, development, responsesto biotic and abiotic environmental stimuli and programmed celldeath (Mittler et al., 2004; Apel and Hirt, 2004; Chung et al.,2008). However, at high concentrations, these ROS can be toxic bydestroying normal metabolism through oxidative damage to lipids,proteins and nucleic acids (Fridovich, 1986). Oxidative damage inthe plant tissue is alleviated by a concerted action of both enzymatic

and non-enzymatic antioxidant mechanism. These mechanismsinclude �-carotens, alpha-tocopherol, ascorbate, glutathione andenzymes including superoxide dismutase (SOD), peroxidase (POX),ascorbate peroxidase (APX), catalase (CAT) and glutathione reduc-tase (GR) (Smirnoff, 1993; Munne-Bosch and Penuelas, 2004). There

4 Exper

ataBad(u

iriCat2ifciÖiteitdttisadefl

2

2

tspoptspasHe

2

aa7

2

od

88 O. Ozkur et al. / Environmental and

re many reports in the literature that underline the intimate rela-ionship between enhanced or constitutive antioxidant enzymectivities and increased resistance to drought stress (Jagtap andhargava, 1995; Türkan et al., 2005). Supporting this idea, enhancedntioxidant defence under drought stress were also reported inrought tolerant plants such as Mediterranean plants such as oakQuercus robur) (Schwanz and Polle, 2001), strawberry tree (Arbutusnedo) and olive tree (Olea europeae) (Sofo et al., 2004).

Caper (Capparis ovata Desf.) which belongs to Cappareacea fam-ly is a winter-deciduous perennial shrub (xerophyte) and droughtesistant plant, grows naturally and flower entirely during summern Mediterranean and semi-arid environments including Greece,yprus and Turkey (Pascual et al., 2008). It can grow in places wherennual rainfall is about 350 mm and easily survive summertimeemperatures higher than 40 ◦C (Barbera, 1991; Söyler and Arslan,000). Beside its use for soil erosion prevention, C. ovata is also

mportant economically since it can be used as drugs, cosmetics andood (Matthaus and Özcan, 2005). Recently, it is widely cultivatedommercially in Morocco, Spain and Italy owing to the increasingnternational demand for its pickled products (Pascual et al., 2008;lmez et al., 2006). It also can resist against high salinity and grow

n poor-nutrient soils. Hence, to elucidate drought related traits ofhis species is not only important to understand how drought tol-rant plants are functioning in their natural environments but alsomprove its agronomic characteristics. However, underlying charac-eristics of physiological and biochemical response of C. ovata to therought is not known. Even, there are no data available related tohe relationship between drought stress and antioxidant defense inhis species. Therefore, in present study, the potential role of antiox-dant enzymes in enhancing its protecting C. ovata from oxidativetress of drought was examined by analyzing enzyme activities suchs SOD, CAT, POX, APX and GR. We also investigated the effects ofrought which is created by application of PEG on the basic param-ters such as growth, relative water content (RWC) and chlorophylluorescence (FV/FM), in leaves of C. ovata.

. Materials and methods

.1. Plant material and treatments

C. ovata seedlings which were obtained from Aegean Agricul-ural Research Institute-Menemen, Izmir (AARI) were used in thistudy. Seedlings were sown into the pots (20 cm × 30 cm) filled witherlite and grown under controlled conditions (light/dark regimef 16/8 h at 27/22 ◦C, relative humidity of 60–70%, photosynthetichoton flux density of (PAR) 350 �mol m−2 s−1). After germina-ion, seedlings were watered regularly with half-strength Hoaglandolution. Polyethylene glycol (PEG) 6000 treatment started whenlants were 35 days old by adding 20% PEG which is equivalent ton osmotic potential of −0.81 MPa, into the half-strength Hoaglandolution. Seedlings were watered regularly with the half-strengthoagland solution containing either 0 or 20% PEG in 2-day intervalsxtended for 14 days.

.2. Growth parameters

Twenty seedlings from each treatment were sampled randomlyt the 0, 7 and 14th days. Fresh weights (g) of shoots were recordednd for dry weight (g) determination samples were oven dried at0 ◦C for 72 h and then weighed.

.3. Relative water content (RWC)

Leaf samples which were collected at the 0, 7 and 14th daysf PEG-treatment were used for RWC assay. After fresh weightetermination, they were floated on deionised water for 5 h under

imental Botany 66 (2009) 487–492

low irradiance. The turgid leaf was quickly blotted dried prior tothe determination of turgid weight. Dry weights of leaves weredetermined after oven-drying at 70 ◦C for 72 h. RWC was calcu-lated according to Smart and Bingham (1974), using the followingformula:

RWC (%) =[

fresh weight − dry weightturgid weight − dry weight

]× 100

2.4. Chlorophyll fluorescence

Leaf of seedlings from six individual plants per treatment wasused for chlorophyll fluorescence analysis. Prior to fluorescencemeasurements, a 1 cm2 circular surface of the upper face of excisedleaf was dark adapted for 30 min using dark leaf clip. The basalnon-variable chlorophyll fluorescence level (F0), the maximal fluo-rescence induction (FM), variable fluorescence (FV) and the ratio ofFV/FM were determined by a Plant Efficiency Analyser (HANSATECHInst. Ltd., Norfolk, UK). More especially, photochemical efficiency ofPS-II (maximum quantum yield FV/FM) was figured.

2.5. Lipid peroxidation

Lipid peroxidation was determined by estimating the TBA-reactive substances (TBARS) content in 0.5 g leaf fresh weightaccording to Madhava Rao and Sresty (2000). TBARS are productsof lipid peroxidation by thiobarbituric acid reaction. The concentra-tion of TBARS (nmol g FW−1) was calculated from the absorbanceat 532 nm (correction was done by subtracting the absorbance at600 nm for unspecific turbidity) by using extinction coefficient of155 mM−1 cm−1.

2.6. Enzyme assays

Fresh leaf samples of both species obtained at 0, 7 and 14 daysafter PEG treatment were used for enzyme analysis. Leaves werefrozen in liquid nitrogen immediately after harvesting and stored at−20 ◦C until enzyme assays. 1 g leaves homogenized in 3 mL 0.05 MNa phosphate buffer (pH 7.8) including 1 mM EDTA and 2% (w/v)PVPP. The homogenate were centrifuged at 13,000 × g for 40 min at4 ◦C. Supernatant was used for enzyme activity and protein contentassays. All assays were done at 4 ◦C. Total soluble protein contents ofthe enzyme extracts were determined according to Bradford (1976)using BSA as a standard. All spectrophotometric analyses were con-ducted on a Shimadzu (UV-1600) spectrophotometer.

Superoxide dismutase (SOD; EC 1.15.1.1) activity assay was basedon the method of Beauchamp and Fridovich (1971) which mea-sures the inhibition in the photochemical reduction of nitrobluetetrazolium (NBT) spectrophotometrically at 560 nm. One unit ofenzyme activity was defined as the quantity of SOD required to pro-duce a 50% inhibition of reduction of NBT and the specific enzymeactivity was expressed as units mg−1 protein. The reaction mix-ture contained 50 mM Na phosphate buffer (pH 7.8), 33 �M NBT,10 mM l-methionine, 0.66 mM EDTA and 0.0033 mM riboflavin.Reactions were carried out at 25 ◦C, under light intensity of about300 �mol−1 m−1 s−1 through 10 min.

Catalase activity (CAT EC 1.11.1.6) was done according toBergmeyer (1970) which measures the decline of the extinction ofH2O2 at the maximum absorption at 240 nm. The reaction mixturecontained 0.05 M Na phosphate buffer (pH 7.0) with 1 mM EDTA andH2O2 (3%). The decrease in the absorption was followed for 3 min

and �mol H2O2 destroyed per min was defined as one unit of CAT.

Ascorbate peroxidase (APX; EC 1.11.1.11) activity was doneaccording to Nakano and Asada (1981). The assay depends onthe decrease in absorbance at 290 nm as ascorbate was oxidized(extinction coefficient of 2.8 mM−1 cm−1). The reaction mixture

Experimental Botany 66 (2009) 487–492 489

c0a

arabcr6d

aiNc53p

i

2

A(drses

3

IPdPtt

(

Focd

Table 1The effect of PEG treatment during experimental period (0, 7 and 14th days) ongrowth parameters shoot fresh and dry weights (g), relative water content (RWC)and photosynthetic efficiency (FV/FM) on Capparis ovata. Means ± SD based on twelvereplicates (n = 12) for fresh weight and dry weight, six replicates (n = 6) for RWC andphotosynthetic efficiency (FV/FM) are presented. Values sharing a common letter arenot significantly different at p < 0.05.

RWC (%) FV/FM Fresh weight (g) Dry weight (g)

Control 0 83.13 ± 2.78b 0.86 ± 0.011ab 0.0872 ± 0.015a 0.0182 ± 0.003b

Control 7 83.71 ± 2.22b 0.86 ± 0.011ab 0.1140 ± 0.005b 0.0202 ± 0.004c

O. Ozkur et al. / Environmental and

ontained 50 mM Na–phosphate buffer (pH 7.0), 0.5 mM ascorbate,.1 mM EDTA Na2 and 1.2 mM H2O2. One enzyme unit was defineds �mol mL−1 oxidized ascorbate per min.

Glutathione reductase (GR; EC 1.6.4.2) activity was measuredccording to Foyer and Halliwell (1976) which depends on theate of decrease in the absorbance of oxidized glutathione (GSSG)t 340 nm. The reaction mixture contained 25 mM Na–phosphateuffer (pH 7.8), 5 mM GSSG, 1.2 mM NADPHNa4. The reaction wasarried out for 3 min and activity of GR was calculated from theeduced GSSG concentration by using the extinction coefficient.2 mM−1 cm−1. One enzyme unit was defined as �mol mL−1 oxi-ized GSSG per min.

Peroxidase activity (POX; EC 1.11.1.7) was based upon the methods described by Herzog and Fahimi (1973) which measures thencrease in absorbance at 465 nm, by the rate of formation of 0.15 Ma–phosphate citrate buffer the oxidized DAB. The reaction mixtureontained DAB solution (dissolved gelatine solution and contained0%, w/v) and 0.6% H2O2. The increase in A465 was followed formin. One enzyme unit was defined as �mol mL−1 destroyed H2O2er min.

The specific enzyme activity for all enzymes was expressed asn unit mg−1 protein.

.7. Statistical analysis

All analyses were done on a completely randomized design.ll data obtained was subjected to one-way analysis of variance

ANOVA) and the mean differences were compared by lowest stan-ard deviations (LSD) test. Each data point was the mean of sixeplicates (n = 6), except for dry and fresh weights of C. ovataeedlings (n = 12). Comparisons with P values <0.05 were consid-red significantly different. In all the figures the spread of values ishown as error bars representing standard errors of the means.

. Results

The level of TBA-reactive substances (TBARS) is given in Fig. 1.n C. ovata, the levels of TBARS showed variation with age and alsoEG treatment. In the control leaves of C. ovata, a small ‘age depen-ent increase’ in TBARS levels became apparent after 14 days of

EG treatment. Where as, TBARS levels significantly increased inhe leaves of drought-stressed C. ovata on day 7 and 14 as comparedo TBARS levels in control groups.

Shoot growth of C. ovata was followed by measuring fresh weightFW) and dry weight (DW) on days 0, 7 and14 (Table 1). Drought cre-

ig. 1. The effect of PEG treatment on lipid peroxidation (MDA content) in leavesf Capparis ovata. Data represents the average of two experiments with three repli-ates. Vertical bars indicate ±SE. Values sharing a common letter are not significantlyifferent at p < 0.05.

PEG 7 75.96 ± 2.44a 0.84 ± 0.006a 0.0898 ± 0.009a 0.0145 ± 0.010a

Control 14 84.02 ± 1.14b 0.85 ± 0.008ab 0.1487 ± 0.012c 0.0220 ± 0.002d

PEG 14 76.29 ± 2.30a 0.83 ± 0.042a 0.1148 ± 0.004b 0.0146 ± 0.003a

ated by application of 20% PEG 6000 significantly decreased freshand dry weights of C. ovata seedlings. Under drought stress, FWdecreased by about 21 and 23%, whereas the decrease in DW wereabout by 28 and 34% on day 7 and 14 of PEG treatments, respectively,as compared to their controls.

The RWC, as indicated by the extent of dehydration were usedto assess cellular damage. Over the experimental period (14 days),RWC in the leaves of C. ovata declined under drought stress (Table 1).However, the results related to the RWC showed similar trend, withabout 8.0% decline on both days 7 and 14 by PEG treatments.

Under drought conditions, a significant alteration in FV/FM wasfound in the leaves of C. ovata (Table 1). As compared to control,FV/FM decreased by 2.90 and 2.59% on day 7 and 14 of PEG treatment,respectively.

In the present study we also observed that drought resultedin higher enzyme activities of SOD, POX, CAT, APX and GR in theleaves of C. ovata. Total SOD activities increased in response to PEGtreatments (Fig. 2). The increases in SOD activity were 1.7-fold and1.8-fold in the leaves of C. ovata on days 7 and 14 of the droughtapplication as compared to their controls. APX activity showed nodifferences in control plants during the experimental period (14days). However, high osmoticum (20% PEG) caused 5 and 4 foldsincrease in APX activity compared to that of the control’s on days 7and 14, respectively (Fig. 2). The increased APX activity observed inthis study might have been due to the increased H2O2 productionunder drought stress. POX activity increased significantly in C. ovataboth on day 7 and 14 under osmotic stress (Fig. 2). The increase hasbeen 2 and 3 folds on day 7 and 14 of the treatment as comparedto controls. PEG treatment result in a significant increase in totalCAT activity in the leaves of C. ovata (Fig. 2). The CAT activity raised1.5 fold on both day 7 and 14 of PEG treatments compared to theircontrols. GR was also significantly increased in drought-stressedseedlings of C. ovata on day 14 and the rate of increment was 3-fold(Fig. 2).

4. Discussion

Drought poses the most important environmental constraintto plant survival and crop productivity in natural and agricul-tural habitats (Chaves et al., 2003). Due to its osmotic effect it caninduce a wide number of responses ranging from growth inhibi-tion and synthesis of some non-toxic compounds to increase theosmotic potential of the cell and thus allow metabolic processesto continue to enhancement of some antioxidant enzyme activities(Türkan et al., 2005). In present study, drought adversely effectedseedling growth of C. ovata as it was evident from decreased dry andfresh weights of the leaves. Inhibition in growth of drought toler-

ant Mediterranean shrubs A. unedo L. (Munne-Bosch and Penuelas,2004) and Atriplex halimus L. (Hassine et al., 2008) were alsoobserved in previous studies. Although C. ovata leaves show somedegree of dessication, it was able to maintain its internal water sta-tus at very similar degree at extended period of drought stress. In

490 O. Ozkur et al. / Environmental and Experimental Botany 66 (2009) 487–492

F es oft not sig

aaowt2

ePAmdo

ig. 2. The effect of PEG treatment on SOD, APX, POX, CAT and GR activities in leavhree replicates. Vertical bars indicate ±SE and values sharing a common letter are

ccordance with this result, Ana Lúcia et al. (2002) also observeddecline in leaf water potential even in drought tolerant clone

f Coffea canephora under water deficit. A decrease in RWC levelsere also observed in A. unedo which can withstand drought condi-

ions of Mediterranean environments (Munne-Bosch and Penuelas,004).

Decrease in stomatal conductance leading to exposure of excessnergy at chloroplasts and over reduction of reaction centers in

SII limits photosynthesis in plants under drought stress (Demmig-dams and Adams, 1992). Photosynthetic efficiency which waseasured as FV/FM values, decreased under drought stress on both

ays as compared to that of non-stressed plants in the leavesf C. ovata. Sofo et al. (2007) also found decreased FV/FM values

C. ovata on days 0, 7 and 14. Data represents the average of two experiments withnificantly different at p < 0.05.

under severely stressed olive plants. Photosynthetic efficiency alsoreduced in the seedlings of 20% PEG treated A. halimus plants(Hassine et al., 2008) and drought-stressed Salvia officinalis (Abreuand Munné-Bosch, 2008). A decrease in photochemical efficiency(FV/FM) is regularly described in temperature and drought stressresponses and may be interpreted as a photo-acclimation process,more than a symptom of damage since it was also the case in otherMediterranean species such as Phyllirea latifolia, A. unedo, S. offici-

nalis and Cistus salvifolius exposed extreme heat-wave of Summer2003 in Southwestern Europe (Munne-Bosch and Penuelas, 2004;Abreu and Munné-Bosch, 2008; García-Plazaola et al., 2008).

Increment in TBA-reactive substances (TBARS) is a good reflec-tion of oxidative damage to membrane lipids and other vital

Exper

miaAlt(m

tthetdrGtc

tHhrIptf(dape

ae(pAdsariatodT

pbaoiaotwti

oecS

O. Ozkur et al. / Environmental and

olecules such as proteins, DNA and RNA. In our study, TBARS levelsncreased in the leaves of C. ovata under drought stress which is ingreement with results of other studies (Schwanz and Polle, 2001;na Lúcia et al., 2002; Sofo et al., 2004). Peroxidation of membrane

ipids may result in enhanced membrane fluidity, which may ledo enhanced electrolyte leakage, as suggested by Thompson et al.1987) and support the hypothesis that drought stress can induce

embrane lipid peroxidation.Under drought conditions, in the electron transport chain, elec-

rons that have no access for their final destination, CO2 are directedo the reduction of molecular oxygen to from superoxide andydrogen peroxide (Miyake and Yokota, 2000). Plants have anfficient system for decomposing reactive oxygen species, usinghe enzymes superoxide dismutase (SOD) and ascorbate peroxi-ase (APX) in chloroplasts (Asada, 1999). In present study, droughtesulted in higher enzyme activities of SOD, POX, CAT, APX andR in the leaves of C. ovata seems to be the physiological adap-

ive mechanisms to regulate its redox status under drought stressonditions.

Among antioxidant enzymes mentioned above, SOD convertshe toxic O2

− to H2O2 which must be scavenged to the O2 and2O by the antioxidant enzymes such as CAT, POX and APX. SOD inigher plants exists in multiple isoforms that are developmentallyegulated and highly reactive to exogenous stimuli (Pan et al., 2006).nduction of total SOD activity which was correlated with increasedrotection from damage associated with oxidative stress implieshat enhancement of SOD scavenge O2

•− radicals to protect C. ovatarom oxidative damage. Similar to our findings Schwanz and Polle2001) found significant increase in SOD levels in water stressed,rought tolerant Mediterranean shrub Q. robur. SOD activity waslso increased by drought stress in shoots of other Mediterraneanlants such as Myrtus communis and Phillyrea angustifolia (Caravacat al., 2005).

APX which is primarily located both in chloroplasts and cytosolcts as a key enzyme of the glutathione–ascorbate pathway. It scav-nges peroxides by converting ascorbic acid to dehydroascorbateNavari-Izzo et al., 1997); it is one of the most important enzymeslaying a vital role in eliminating toxic H2O2 from plant cell insada–Halliwell pathway (Foyer et al., 1994). In the present study,rought stress increased total APX activity differed prominently intressed leaves as compared to that of non-stressed leaves. Higherctivity of APX in stressed leaves suggests a more effective H2O2emoval which might be produced by an enhanced activity of SODn C. ovata under drought conditions. A higher APX activity waslso reported in tobacco BY-2 cell cultures (Bueno et al., 1998) andepary bean under drought stress (Türkan et al., 2005). Parallel tour results, an upregulation in APX activity was also observed inrought-stressed olive (Sofo et al., 2007) and maize (Pastori andrippi, 1992).

POX is among the major enzymes that scavenge H2O2 in chloro-lasts which are produced through dismutation of O2

− catalyzedy SOD (Asada and Takahashi, 1987). In tolerant plant species, POXctivity was found to be higher, providing protection against thexidative stress (Scalet et al., 1995). Remarkably higher levels of POXn drought-stressed seedlings of C. ovata might also be considereds a higher capacity to decompose H2O2 more rapidly. Supportingur results, Yang et al. (2008) also reported induced higher activi-ies in Picea aspertata seedlings under drought stress supplementedith a high light. In one of our previous studies we also observed

hat drought resistant tepary bean showed a higher constitutive andnduced activity of POX under drought stress (Türkan et al., 2005).

CAT eliminates H2O2 by breaking it down directly to water andxygen (Tsang et al., 1991). In our study, total CAT activity wasnhanced by drought in the leaves of C. ovata. These results areonsistent with the reports from Mittler and Zilinkas (1993) andofo et al. (2007) who found that with drought stress, CAT activities

imental Botany 66 (2009) 487–492 491

increased in drought-stressed peas and olive, respectively. Whenone considers that CAT acts in peroxisomes, it is logical to suggestthat photorespiration in the leaves of C. ovata was also affected byPEG treatment.

In the present study, drought stress result in a significantincrease in GR the activity which is of another major enzyme thathas a role in the H2O2 scavenging in Asada–Halliwell pathwayin plant cells (Noctor et al., 2002). This might be due to main-tain a high ratio of NADP+/NADPH, therefore ensuing availabilityof NADP+ to accept electrons from photosynthetic electron trans-port chain and to facilitate the regeneration of oxidized ascorbate(Noctor et al., 2002; Yang et al., 2008). We could conclude thatascorbate–glutathione cycle efficiently eliminates the deleteriouseffects of reactive oxygen species in the leaves of C. ovata underdrought stress. Drought induced increase in GR was also observedin Picea asperata seedlings under highlight conditions. Parallel toour results GR activity significantly increased in drought tolerantmaize strain under water scarcity (Pastori and Trippi, 1992).

5. Conclusion

In conclusion, as a whole, the antioxidant system of C. ovata func-tioned at higher rates to restrain an increased ROS formation underdrought. This seemed to be evident by evaluating the extent of cellu-lar damages, which was less remarkable under stress. These resultsare in good agreement with several studies indicating that droughtgenerally induces antioxidative system in Mediterranean plants andincreased drought tolerance is correlated with diminishing oxida-tive injury (Pastori and Trippi, 1992; Smirnoff, 1993; Polle, 1997;Schwanz and Polle, 2001).

Acknowledgment

This work was supported by grant-in-aids 2008-FEN-039 fromEge University Research Foundation.

References

Abreu, M.E., Munné-Bosch, S., 2008. Salicylic acid may be involved in the regulationof drought-induced leaf senescence in perennials: a case study in field-grownSalvia officinalis L. plants. Environ. Exp. Bot. 64 (2), 105–112.

Ana Lúcia, S., Lima, F., DaMatta, M., Hugo, A.P., Marcos, R.T., Marcelo, E., 2002. Photo-chemical responses and oxidative stress in two clones of Coffea canephora underwater deficit conditions. Environ. Exp. Bot. 47 (3), 239–247.

Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress andsignal transduction. Annu. Rev. Plant Biol. 55, 373–399.

Asada, K., Takahashi, M., 1987. Production and scavenging of active oxygen in pho-tosynthesis. In: Kyle, D.J., Osmond, C.B., Arntzen, C.J. (Eds.), Photoinhibition.Elsevier, Amsterdam, pp. 227–287.

Asada, K., 1999. The water–water cycle in chloroplasts: scavenging of active oxygensand dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50,601–639.

Barbera, G., 1991. Programme de Recherché Agrimed le Caprier (Capparis spp.) Com-mission des Communautes Europeannes. Serie Agriculture EUR, Luxenburg, p.13617.

Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and appli-cable to acrylamide gels. Anal. Biochem. 44, 276–287.

Bergmeyer, N., 1970. Methoden der Enzymatischen Analyse, vol. 1. Akademie Verlag,Berlin, pp. 636–647.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. Anal.Biochem. 72, 248–254.

Bueno, P., Piqueras, A., Kurepa, J., Savoure, A., Verburggen, N., Van Montagu, M.,Inze, D., 1998. Expression of antioxidant enzymes in response to ABA and highosmoticum in tobacco BY-2 cell cultures. Plant Sci. 138, 27–34.

Caravaca, F., Alguacil, M.M., Hernandez, J.A., Roldan, A., 2005. Involvement of antiox-idant enzyme and nitrate reductase activities during water stress and recoveryof mycorrhizal Myrtus communis and Phillyrea angustifolia plants. Plant Sci. 169,

191–197.

Chaves, M.M., Maroco, J.P., Pereira, J.S., 2003. Understanding plant responses todrought-from genes to the whole plant. Funct. Plant Biol. 30, 239–264.

Chung, J.S., Zhu, J.K., Bressan, R.A., Hasegawa, P.M., Shi, H., 2008. Reactive oxygenspecies mediate Na+-induced SOS1 mRNA stability in Arabidopsis. Plant J. 53,554–565.

4 Exper

D

F

F

F

G

H

H

J

M

M

M

M

M

M

N

N

N

92 O. Ozkur et al. / Environmental and

emmig-Adams, B., Adams, W.W., 1992. Photoprotection and other responses ofplants to high light stress. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 599–626.

oyer, C.H., Halliwell, B., 1976. Presence of glutathione and glutathione reductase inchloroplasts: a proposed role in ascorbic acid metabolism. Planta 133, 21–25.

oyer, C.H., Lelandais, M., Kunert, K.J., 1994. Photooxidative stress in plants. Physiol.Plant 92, 696–717.

ridovich, I., 1986. Biological effects of superoxide radical. Arch. Biochem. Biophys.247, 1–11.

arcía-Plazaola, J.I., Esteban, R., Hormaetxe, K., Fernández-Marín, B., María Becerril,J.M., 2008. Photoprotective responses of Mediterranean and Atlantic trees tothe extreme heat-wave of summer 2003 in Southwestern Europe. Trees-Struct.Funct. 22 (3), 385–392.

assine, A.B., Ghanem, M.E., Bouzi, S., Lutts, S., 2008. An inland and a coastal pop-ulation of Mediterranean xero-halophyte species Atriplex halimus L. differ intheir ability to accumulate proline and glycinebetaine in response to salinityand water stress. J. Exp. Bot. 59, 1315–1326.

erzog, V., Fahimi, H., 1973. Determination of the activity of peroxidase. Anal.Biochem. 55, 554–562.

agtap, V., Bhargava, S., 1995. Variation in the antioxidant metabolism of drought tol-erant and drought susceptible varieties of Sorghum bicolor (L.) Moench. exposedto high light, low water and high temperature stress. J. Plant Physiol. 147,195–197.

adhava Rao, K.V., Sresty, T.V.S., 2000. Antioxidative parameters in the seedlings ofpigeonpea (Cajanus cajan L. Millspaugh) in response to Zn and Ni stresses. PlantSci. 157, 113–128.

atthaus, B., Özcan, M., 2005. Glucosinolates and fatty acid, sterol and tocopherolcomposition of seed oils from Capparis spinosa var spinosa and Capparis ovataDesf. Var canescens Heywood. J. Agric. Food Chem. 53, 7136–7141.

ittler, R., Vanderauwera, S., Gollery, M., Van Breusegem, F., 2004. Reactive oxygengene network of plants. Trends Plant Sci. 9 (10), 490–498.

ittler, R., Zilinkas, B., 1993. Detection of ascorbate peroxidase activity in native gelsby inhibition of the ascorbate dependent reduction of nitroblue tetrazolium.Anal. Biochem. 212, 540–546.

iyake, C., Yokota, A., 2000. Determination of the rate of photoreduction of O2 inthe water-water cycle in watermelon leaves and enhancement of the rate bylimitation of photosynthesis. Plant Cell Physiol. 41, 335–343.

unne-Bosch, S., Penuelas, J., 2004. Drought induced oxidative stress in strawberrytree (Arbutus unedo L.) growing in Mediterranean field conditions. Plant Sci. 166,1105–1110.

akano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate specificperoxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880.

avari-Izzo, F., Meneguzzo, S., Loggini, B., Vazzana, C., Sgherri, C.L.M., 1997. The roleof the glutathione system during dehydration of Boea hygroscopica. Physiol. Plant99, 23–30.

octor, G., Gomez, L., Vanacker, H., Foyer, C.H., 2002. Interactions between biosynthe-sis, compartmentation and transport in the control of glutathione homeostasisand signalling. J. Exp. Bot. 53, 1283–1304.

imental Botany 66 (2009) 487–492

Ölmez, Z., Göktürk, A., Özalp, M., 2006. Determining growth of Caper (Capparis ovataDesf.) plantations with eleven different provenances on an erosion control areain Turkey. Pakistan J. Biol. Sci. 9 (5), 880–884.

Pan, Y., Jun Wu, L., Liang Yu, Z., 2006. Effect of salt and drought stress on antioxidantenzyme activities and SOD isoenzymes of liquorice (Glycyrrhiza uralensis Fisch).Plant Growth Regul. 49, 157–165.

Pascual, B., San Bautista, A., Lopez-Galarza, S., Alagarda, J., Maroto, J.V., 2008. Intactfruit of Caper (Capparis spinosa) is an improved seed propagation method. ActaHortic. 782, 107–114.

Pastori, G., Trippi, V., 1992. Oxidative stress induced high rate of glutathionereductase synthesis in a drought resistant maize strain. Plant Cell Physiol. 33,957–961.

Polle, A., 1997. Defence against photooxidative damage in plants. In: Scandalios, J.(Ed.), Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Har-bour Laboratory Press, Cold Spring, pp. 623–666.

Rhizopoulou, S., Psaras, G.K., 2003. Development and structure of drought tolerantleaves of the Mediterranean shrub Capparis spinosa L. Ann. Bot. 92, 377–383.

Scalet, M., Federice, R., Guido, M.C., Manes, F., 1995. Peroxidase activity andpolyamine changes in response to ozone and simulated acid rain in Aleppo pineneedless. Environ. Exp. Bot. 35, 417–425.

Schwanz, P., Polle, A., 2001. Differential stress responses of antioxidative system todrought in Quercus robur and Pinus pinaster grown under high CO2 concentra-tions. J. Exp. Bot. 52 (354), 133–143.

Smart, R.E., Bingham, G.E., 1974. Rapid estimates of relative water content. PlantPhysiol. 53, 258–260.

Smirnoff, N., 1993. The role of active oxygen in response of plants to water deficitand desiccation. New Phytol. 125, 27–58.

Sofo, A., Dichio, B., Xiloyannis, C., Masia, A., 2004. Effects of different irradiancelevels on some antioxidant enzymes and on malondialdehyde content duringrewatering in olive tree. Plant Sci. 166, 293–302.

Sofo, A., Manfreda, S., Dichio, B., Florentino, M., Xiloyannis, C., 2007. The olive tree:a paradigm for drought tolerance in Mediterranean climates. Hydrol. Earth Syst.Sci. Disc. 4, 2811–2835.

Söyler, D., Arslan, N., 2000. The effects of some plant growth regulators on the rootingof Caper (Capparis spinosa L.). Turk. J. Agric. For. 24, 560–595.

Thompson, J.E., Legge, R.L., Barber, R.L., 1987. The role of free radicals in senescenceand wounding. New Phytol. 105, 317–334.

Tsang, E.W.T., Bowler, C., Herouart, D., Van Camp, W., Villarroel, R., Genetello, C., VanMontagu, M., Inzé, M., 1991. Differential regulation of superoxide dismutases inplants exposed to environmental stress. Plant Cell 3, 783–792.

Türkan, I., Bor, M., Ozdemir, F., Koca, H., 2005. Differential responses of lipid peroxi-

dation and antioxidants in the leaves of drought tolerant P. acutifolius Gray anddrought sensitive P. vulgaris L. subjected to PEG mediated water stress. Plant Sci.168, 223–231.

Yang, Y., Han, C., Liu, Q., Lin, B., Wang, J., 2008. Effect of drought and low lighton growth and enzymatic antioxidant system of Picea asperata seedlings. ActaPhysiol. Plant. 30, 433–440.