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Plant Physiology and Biochemistry 61 (2012) 108e114

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Effect of water withdrawal on formation of free radical, proline accumulation andactivities of antioxidant enzymes in ZAT12-transformed transgenic tomato plants

Avinash Chandra Rai a,b, Major Singh a, Kavita Shah c,*

aDivision of Vegetable Improvement, Indian Institute of Vegetable Research, Shahanshahpur, Jhakhini, Varanasi, U.P. 221305, IndiabDepartment of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, U.P. 221005, Indiac Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi, U.P. 221005, India

a r t i c l e i n f o

Article history:Received 10 August 2012Accepted 14 September 2012Available online 18 October 2012

Keywords:AntioxidantProlineSolanum lycopersicumTomatoZAT12Zinc finger protein

Abbreviations: APX, ascorbate peroxidase; CATreductase; POD, guaiacol peroxidase; NT, non-transfspecies; SOD, superoxide dismutase; T, transformed.* Corresponding author. Tel.: þ91 542 6701663,

fax: þ91 542 2307225.E-mail addresses: kshah.iesdbhu@sify.com, csshah

0981-9428/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.09.010

a b s t r a c t

Water stress often leads to the accumulation of reactive oxygen species (ROS) and their excessiveproduction alters the activities of enzymes involved in their removal. ZAT12 is a member of stress-responsive C2H2 type Zinc Finger Protein (ZFP) reported to control the expression of severalstress-activated genes in plants through ROS signaling. The ZAT12-transformed tomato lines (cv. H-86variety Kashi Vishesh) when subjected to water withdrawal for 7, 14 and 21 days revealed significant andconsistent changes in activities of enzymes SOD, CAT, APX, GR and POD paralleled with an increasedproline levels. Unlike that in wild-type tomato, the leaf superoxide anion and hydrogen peroxideconcentrations in the transformed tomato plants did not alter much, suggesting a well regulatedformation of free radicals suppressing oxidative stress in the latter. Results suggest BcZAT12-transformedtomato lines ZT1, ZT2 and ZT6 to be better adapted to drought stress tolerance by accumulation ofosmolyte proline and increased antioxidant response triggered by the ZAT12 gene. Therefore, theZAT12-transformed tomato cv. H-86 lines will prove useful for higher yield of tomato crop in regionsaffected with severe drought stress.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Abiotic stresses lead to specific genetic responses therebyresulting in an altered gene expression and their translationproducts in plants to help them adapt to the environment [1].Water deficit is one of the major environmental factors that lead toa substantial loss of crop yield [2]. Statistical data suggest thatpercentage of drought affected land area has doubled from the 1970to the early 2000 worldwide [3]. Water scarcity often leads to theexcess production and accumulation of reactive oxygen species(ROS) inhibiting the photochemical behavior and altering theactivities of enzymes involved and photosynthetic activity [4].

Formation of ROS viewed as a threat to the cell is either byelectron leakage from the electron transport activities or asa byproduct of various metabolic pathways localized in differentcellular compartments [5]. ROS are involved in stress-signal

, catalase; GR, glutathioneormed; ROS, reactive oxygen

þ91 9450955423 (mobile);

in@yahoo.co.in (K. Shah).

son SAS. All rights reserved.

transduction pathway, may cause lipid peroxidation and subse-quent membrane injury as well as protein and nucleic acid damage[6]. The production of ROS under normal growth conditions in cellsis low (w240 mMs�1 O2

��) and in chloroplasts a steady state(w0.5 mM H2O2) is maintained, however, an enhanced productionof ROS under environmental stress results in altered cellularhomeostasis (upto 720 mMs�1 O2

��) and a steady state of 5e15 mMH2O2 in chloroplasts [5]. To prevent these damages, plants containa range of protective and repair systems, which under normalcircumstances, minimize the occurrence of oxidative damage. Thereare systems which either react with reactive forms of oxygen andkeep them at a low level or the antioxidant enzymes as superoxidedismutase (SOD), catalase (CAT), ascorbate peroxidase (APX),glutathione reductase (GR) and guaiacol peroxidase (POD) thatquench ROS supported by antioxidants like glutathione, ascorbicacid, a-tocopherol and carotenoids or the enzyme POD that main-tains cellular balance of ROS [1,6,7]. Once SOD converts superoxideradical to H2O2, it is reduced to water and oxygen either by APX inascorbateeglutathione cycle or by POD and CAT in cytoplasm and inother cellular compartments [8].

Proline is a powerful indicator of drought stress and itsaccumulation together with other soluble nitrogenous compoundsunder water deficit conditions in plants is regarded as an important

Fig. 2. Hydrogenperoxide formation in transformed (ZT1-ZT6) andnon-transformed (NT)tomato leaves at increasingdays ofwaterwithdrawal. Thedata aremeanof three replicates�SD. ANOVA significant at P� 0.01; (*) represent values significantly different at P< 0.05.

A. Chandra Rai et al. / Plant Physiology and Biochemistry 61 (2012) 108e114 109

strategy adopted by plants to prevent cellular dehydration bybalancing the osmotic strength of the cytoplasm with that of theenvironment [9] or a powerful scavenger of �OH in plants func-tioning thereby as an antioxidant [10].

Tomato (Solanum lycopersicum) is an economically importantvegetable of the Solanaceae family, consumed globally [11]. Tomatogrowth and productivity is often challenged by various abiotic andbiotic stress factors [12]. To overcome this threat, plant geneticengineering comes to rescue by providing an emphatic tool tointroduce the biotic and abiotic stress-responsive genes in thedesired organism for stress tolerance. ZAT12 is a member of stress-responsive C2H2 type Zinc Finger Protein (ZFP) reported to controlthe expression of several stress-activated genes in plants throughROS signaling [13,14]. Earlier we successfully carried out Agro-bacteriummediated transformation of the tomato cv. H-86 (v. KashiVishesh), carrying ZAT12 gene of Brassica carinata homologous toArabidopsis ZAT12 (accession no. DQ166621.1) reported in databasesunder the control of Bclea1 promoter to obtain drought toleranttransgenic tomato (Fig. 1). We obtained six ZAT12-transformedtomato lines ZT1-ZT6 having an increased number of ZAT12 tran-scripts under drought [41]. The present study was undertaken withtheobjective to examine theeffect ofwaterwithdrawal on formationof ROS and changes in expression of enzymes SOD, APX, CAT, GR andPOD in ZAT12-transformed (T) tomato lines as compared to non-transformed (NT)wild-type. As the engineeringof ZAT12 in tomato isalso likely to improve plant tolerance involving osmoprotectants, thepresent studyalso examines the level of proline in transgenic tomatolines subjected to drought.

2. Results

2.1. Effect of increasing days of water withdrawal on hydrogenperoxide formation

The effect of increasing days of water withdrawal stress onformation of hydrogen peroxide in non-transformed (NT) andtransformed (T) plants (Fig. 2) suggest higher values of H2O2 underdrought conditions that increased gradually with increasing days ofstress in both NT and T plants. The NT plants however had signif-icantly higher 2.1e2.4 times H2O2 levels during 14e21 days ofwater withdrawal when compared with T tomato lines, whereonly 1.2e1.9 times increase in H2O2 were noted. Earlier we noted

Fig. 1. ZAT12-transformed tomato plant showing tolerance upon 21 days of waterwithdrawal compared to non-transformed (NT) control plants.

that the expression level of ZAT12 transcripts increased uponexposure to drought stress in ZAT12-transformed tomato lines [41].

2.2. Effect of increasing days of water withdrawal on formation ofsuperoxide anion

Increasing days of water withdrawal show NT plants to havehigher superoxide anion formation (Fig. 3) under both non stress (0days) as well as drought conditions. Lines ZT4 and ZT6 revealeda significant suppression in formation of superoxide anion (23%) ascompared to NT plants. At day 7 a maximum of 103% highersuperoxide anion were recorded in NT plants as compared with Tlines. In contrast to NT plants, beyond 7 days of water stress, thetransformed tomato lines always had similar and significantlylowered superoxide anion formation with almost no change invalues at 14 and 21 days of drought. In ZT1 tomato line thesuperoxide anion levels remained almost unchanged throughoutthe drought stress period (Fig. 3).

2.3. Superoxide dismutase (SOD) activity

A gradual increase in the activity of SOD were observed in thetransformed tomato lines with increasing days of water deficitcondition (Fig. 4). At 7 day of water withdrawal a 44% increase in

Fig. 3. Superoxide (O2��) anion formation in ZT and NT tomato lines (refer to Fig. 2).

Fig. 4. Superoxide dismutase activity in ZT and NT tomato lines (refer to Fig. 2). Fig. 6. Ascorbate peroxidase activity in ZT and NT tomato lines (refer to Fig. 2).

A. Chandra Rai et al. / Plant Physiology and Biochemistry 61 (2012) 108e114110

SOD activity were noted in ZT6 tomato line which increasedthereafter to w62% at 14 and 21 days of water withdrawal whencompared to NT plants. Among T tomato lines ZT6 > ZT2 > ZT1exhibited maximum SOD activity in plants grown under 14 and 21days of drought conditions (Fig. 4).

2.4. Catalase (CAT) activity

The activity of CAT in general increased in both NT and T plantsunder non stress (0 day) as well as upon 7e21 days of waterwithdrawal however, the increase was always significantly higherin T plants as compared to NT plants (Fig. 5). Maximum CAT activitywere noted in ZT1 > ZT4 > ZT2 tomato lines when subjected to 7e21 days of drought compared with NT tomato plant. Line ZT1 hadsignificant elevation of 48% in CAT activity at 7 days of water stresscondition.

2.5. Ascorbate peroxidase (APX) activity

The specific activity of APX under increasing days of waterwithdrawal (Fig. 6) suggests a significant elevation in APX activityin all T tomato lines. At 7 days of drought stress ZT2 had 63% APXactivity when compared with NT plants. The T lines ZT1, ZT2 andZT6 at 14 days of water withdrawal had APX activity of 68%, 74%,and 70%, respectively, whereas beyond 21days the activity

Fig. 5. Catalase activity in ZT and NT tomato lines (refer to Fig. 2).

increased significantly to 82% in ZT1-ZT2 and 79% in ZT6 respec-tively, as and when compared to NT tomato plants. In 21 daysdrought stressed tomato plants the APX activity in T lines were inthe order ZT1 > ZT2 > ZT6 > ZT5 > ZT4 > ZT3.

2.6. Glutathione reductase (GR) activity

Similar to POD activity the activity of enzyme GR in T and NTtomato lines increased significantly during 7e14 days of droughtwith a gradual decline thereafter (Fig. 7). Controls from transgenictomato had higher GR specific activity compared with wild type. At7 days of water stress the T tomato lines ZT1-ZT6 had almost similaror slight increase in GR activity compared with NT tomato lineswhereas at day 14 of water withdrawal a significant elevation ofw80% in GR activity in transgenics were noted as compared withNT lines. MaximumGR activity of 76%, 96% and 69%were associatedwith transgenic lines ZT2, ZT3 and ZT6 respectively, at 14 day ofwater stress than the non stressed NT plants (Fig. 7).

2.7. Guaiacol peroxidase (POD) activity

The POD activity in both NTand T plants increased initially till 14days of drought and decreased thereafter (Fig. 7). Under non stressconditions the activity of POD in T tomato lines (ZT1eZT6) were

Fig. 7. Glutathione reductase activity in ZT and NT tomato lines (refer to Fig. 2).

Fig. 8. Guaiacol peroxidase activity in ZT and NT tomato lines (refer to Fig. 2).

A. Chandra Rai et al. / Plant Physiology and Biochemistry 61 (2012) 108e114 111

higher compared to POD activity in control NT plants (Fig. 8). At day7 of water withdrawal the POD activity in T lines increased almost70% to that in NT plants with a subsequently significant increase of89% at day 14 in transgenic tomato. At 7 days of water withdrawalthe line ZT6 had a maximum POD activity of 77% comparedwith NTplants, whereas at day 14, ZT1 showed a significantly higher PODactivity of w90% as and when compared with NT tomato plants(Fig. 8).

2.8. Accumulation of proline (Pro) under drought

The amount of Pro in both NT and T tomato plants increasedwith increasing days of drought stress (Fig. 9). The proline levels inthe T lines were in order ZT2 > ZT6 > ZT1 � ZT4 > ZT5 > ZT3 > NT.Tomato line ZT2 accumulated 2.5 times more proline followed by2e1.9 times in ZT6 and ZT1 plants than the corresponding controlNT plants.

3. Discussion

Growth reduction under water stress condition is a major risk intomato yield [15]. It is the plant water relation that plays a key rolein the activation and intonation of antioxidant defense mechanismunder drought [16]. There is an increasing evidence that suggestsoxidative stress to be the key damaging factor in plants exposed tostressful conditions [1]. Drought induced reduction in leaf

Fig. 9. Proline content in ZT and NT tomato lines (refer to Fig. 2).

pigments is considered to be a typical oxidative stress indicatorwhich might be attributed to pigment photo-oxidation, chlorophylldegradation or deficiency in chlorophyll synthesis [17]. To over-come ROS mediated oxidative stress, plants employ primarilyantioxidant enzymes SOD, CAT, APX and GR [6,10]. There are manyreports of the production of abiotic stress tolerant transgenic plantsby cloning and upregulating stress-inducible genes that in turnresult in over-expression of antioxidant enzymes [10] and accu-mulation of compatible solutes [9,18] which confers tolerance toplants toward that particular stress factor. The transformed tomatoplants harboring, BcZAT12, a stress-inducible gene is quite likely toalter the expression of antioxidant enzymes as well as the level ofproline, thereby providing tolerance toward drought stress totransgenic tomato plants. Therefore, the present study has beencarried out to develop an understanding toward induced oxidativestress, antioxidant defense and role of proline in wild type andtransgenic tomato lines (ZT1eZT6) cv. H-86 var. Kashi Visheshwhen exposed for 7, 14 and 21 days of drought conditions. Resultsindicate reduction in oxidative stress paralleled with alteredactivities of SOD, APX, GR, CAT and POD and enhanced prolinelevels in transgenic tomato lines ZT1, ZT6 and ZT4 in particular ascompared to non-transformed wild-type counterparts.

The H2O2 concentration was measured in leaves as an indicatorof oxidative stress in plants [19]. Water deficit raised the concen-tration of H2O2 in wild-type tomato. The H2O2 concentrationssignificantly lowered in transformed plants as compared with NTplants, suggesting a lowered accumulation of H2O2 in BcZAT12transformants, indicating thereby a balanced H2O2 formation in thetransformed tomato lines.

Superoxide anion (O2��) is the first ROS produced from uptake of

electron of molecular O2 passing through the photosystem. In ourexperiments the level of O2

�� observed with increasing days of waterwithdrawal in NT and T lines appear to be function of time andincreasing stress. The production of O2

�� has been shown to bedependent upon the intensity of stress, repeated stress periods, ageand species of the plants [6,20,21]. Decrease in H2O2 and super-oxide anion formation is suggestive of improved cellular homeo-stasis of ROS in transgenic tomato plants.

Altered antioxidant enzyme activities facilitate the adjustmentrequired to overcome, avoid or neutralize stress [6]. MetalloenzymeSOD removes O2

�� by catalyzing its dismutation, where one O2�� is

reduced to H2O2 and another oxidized to O2 [1,10,22,23]. Activity ofCAT is reported to enhance under severe drought stress whereasunder moderate drought stress the H2O2 scavenging is preferablymade by ascorbic acid through the ascorbate/glutathione cycle[24e28].

Drought tolerant plants often have higher POD activity thansensitive plants under drought stress conditions as reported indrought tolerant common bean, sunflower and sorghum [29e31].In accordance with these reports an increase in SOD, CAT and PODactivities in T tomato lines ZT1, ZT2 and ZT6 at increasing days ofwater withdrawal compared with NT lines were also noted in ourresults.

It is assumed that the increased activities of the enzymes ofascorbate/glutathione pathway, especially that of APX confergeneral resistance to array of environmental stresses [20,27,32]. Inour result the expression of APX in T tomato lines increasedcontinuously at all days (7,14 and 21) of water stress comparedwithNT line. The T lines ZT1, ZT2 and ZT6 showed the significant eleva-tion of APX activities during 14 as well as 21 days of water stresssuggesting these lines to be more adapted to drought conditions.

The GR enzyme is responsible for converting the oxidizedglutathione disulfide (GSSG) to glutathione (GSH) using NADPH[32] and not only it keeps glutathione in the reduced state but it isalso responsible for the maintenance of the cellular GSH:GSSG

A. Chandra Rai et al. / Plant Physiology and Biochemistry 61 (2012) 108e114112

ratio. Changes in H2O2 levels affects this ratio and the interactionbetween H2O2/GSH/GR redox system helps combat abiotic stressesin plants [1,33e35]. With the imposition of drought for 7e14 days,the T tomato plants had significant increase of GR activitycompared with NT line. This could possibly be a result of changes inexpression of gene, regulating its synthesis. It indicates that Ttomato lines are more capable of drought resistance than NT plants.

Among various compatible solutes, proline (Pro) is known tohave various functions under stress conditions in plants [10]. Itserves as a mediator of osmotic adjustment, a stabilizer of subcel-lular insert structure, an eliminator of free radicals, a buffer of redoxpotential and an important component of cell wall proteins [36,37].Therefore, Pro is now regarded as a non enzymatic antioxidant thatmicrobes, animals and plants require tomitigate the adverse effectsof ROS [10]. It is well documented that there is a dramatic accu-mulation of Pro following salt, drought and metal stress due to itsincreased synthesis or decreased degradation [9,10]. An increasedaccumulation of Pro observed in transgenic tomato plants in ourstudy during 7e14 days of drought period can therefore be corre-lated with improved tolerance to drought stress.

The antioxidant enzymes SOD and CAT together with enzymePOD function as effective quenchers of ROS and their level may alsodetermine the sensitivity of plants to lipid peroxidation [6,20]. It hasalso been elicited that POD accounts for w50% of H2O2 productionduring oxidative burst in Arabidopsis [39]. Apart from H2O2

mitigation POD also functions in cell-growth and lignin formationinvolving several different substrates in plants [20,38,39].Therefore, POD alone cannot be accounted for plant defense understress as it does not have the same function as APX or CAT. Presentresults showed that at 7 and 14 days of stress, the activitiesof enzymes SOD, CAT and POD were high. CAT had very moderateactivity in T tomato lines but the level of SOD showed an increaseduring the stress period. In our experiments a higher APX activityin 21 days stressed T tomato lines, therefore suggest that theexcess ROS (H2O2) formed from SOD during the severe droughtstress conditions was possibly detoxified by APX enzyme viaHalliwelleAsada cycle.

Ectopic over-expression of SOD activity relative to the H2O2scavenging activity is reported to be detrimental to cells in variousorganisms [6]. The activity of SOD at times may not be high enoughto scavenge all O2

�� butmay be sufficient to producemore H2O2 thanin controls resulting inmore formation of reactive hydroxyl radicalsinvolving remaining O2

�� and H2O2. Therefore, it is the increase inthe ratio of O2

�� scavenging enzyme SOD to H2O2 scavengingenzymes CAT/APX activity (Table 1) rather than individual changesin the activity of each enzymes that would cause and decide theextent of oxidative stress [1,10,20]. In our study, mostly the ratioSOD:CAT or SOD:APX activity increased during 14e21 days droughtstress, in non-transformed tomato plants. In transformed tomatolines however, the ratio of SOD:APX decreased with a reciprocaltrend for SOD:CAT activity which increased with increase indrought stress. These enzymatic alterations strengthen the fact that

Table 1Comparison of activity ratios of antioxidant enzymes at 10 leaf stage plants of tomato cv

Genotypes Drought stress (days)

Control 7

SOD/CAT SOD/APX SOD/CAT SOD/A

NT 0.347 0.180 0.239 0.080ZT1 0.331 0.060 0.209 0.080ZT2 0.265 0.070 0.288 0.120ZT3 0.169 0.110 0.114 0.090ZT4 0.381 0.030 0.402 0.080ZT5 0.191 0.150 0.241 0.120ZT6 0.321 0.080 0.520 0.090

drought induced oxidative stress in tomato plants is regulatedprimarily by the enzymes SOD, APX and CAT in transgenic tomatoplants, thereby helping them to better adapt to drought conditions.

In summary, the transgenic tomato plants had an altered anti-oxidant enzyme activities under drought. An increase in SOD, CAT,APX and GR activities appearedwith a higher proline accumulation.The transformed tomato plants did not show stress since leaf O2

��

and H2O2 concentrations did not alter much, however when thenon-transformed control plants subjected to drought were studied,the antioxidant enzymes activities were more limited and theoxidative stress was evident. These results suggest that ZAT12-transformed tomato plants especially ZT1, ZT2 and ZT6 are betteradapted to drought and can withstand water withdrawal due toimproved antioxidant response as well as accumulation of osmo-lyte proline, making these ZAT12 transgenics of tomato useful forimproving the yield in water stressed area. Trial’s using transgenictomato lines for yield under water scarcity is presently in progress.

4. Materials and methods

4.1. Plant material and stress conditions

Seeds from six transformed tomato lines (cv. H-86 v. KashiVishesh) harboring ZAT12 gene developed earlier in our lab andconfirmed for integration of ZAT12 gene in tomato plants [41] weresown and raised inside a net house. Seedlings of all the six lineswere tested by kanamycin spray for the selection of true transgenicplants and raised to ten leaves stage in a net house. The droughttreatments began at 10 leaf stage and the control non-transformed(NT) and transformed (T) plants were maintained for 21 days(Fig. 1). The control plants received 100% field capacity irrigation.The leaves from the controls as well as drought stressed transgenictomato plants at 7, 14 and 21 days were collected and used forexperiments. All the estimations were carried out in triplicate.

4.2. Hydrogen peroxide (H2O2) measurement

The H2O2 levels in leaf samples fromNTand T tomato plants at 7,14 and 21 days of drought stress as earlier [6]. For extraction ofH2O2 about 200 mg tissue was homogenized in 5 ml of 50 mMphosphate buffer (pH 6.5). The homogenate was centrifuged at7000 � g for 20 min. To 3 ml of the supernatant, 1 ml of 0.1% tita-nium sulphate in 20% H2SO4 was added. The mixture was thencentrifuged at 7000� g for 15 min. The intensity of the yellow colordeveloped was measured at 410 nm. The amount of H2O2 wascalculated using an extinction coefficient of 0.28 mM�1 cm�1 andexpressed as mmol g�1 fresh wt.

4.3. Superoxide anion (O2��) measurement

The rate of superoxide (O2��) anion production was measured

according to Misra and Fridovich [22]. About 200 mg fresh plant

. H-86 (Kashi Vishesh) after 7, 14 and 21 days of water withdrawal.

14 21

PX SOD/CAT SOD/APX SOD/CAT SOD/APX

0.221 0.030 0.208 0.0300.358 0.070 0.329 0.0600.235 0.070 0.328 0.0700.207 0.060 0.235 0.0600.316 0.070 0.454 0.0600.250 0.070 0.297 0.0600.463 0.090 0.453 0.060

A. Chandra Rai et al. / Plant Physiology and Biochemistry 61 (2012) 108e114 113

samples were homogenized using cold mortar and pestle in cold(0e4 �C), in 50 mM potassiumephosphate buffer (pH 7.5) con-taining 250 mM sucrose, 10 mM MgCl2 and 1 mM diethyl dithio-carbamate to inhibit SOD activity. After centrifugation at 22,000� gfor 20 min, superoxide anion (O2

��) was measured in the superna-tant, by its capacity to reduce epinephrine. The assay mixture intotal volume of 3 ml containing 3 mM epinephrine in phosphatebuffer (pH 7.5), 0.3 mMNADH and the supernatant. The absorbancewas recorded at 480 nm in an Elico SL-159, (India) UVeVis spec-trophotometer and NADH dependent adrenochrome formationwasrecorded for 7e8 min. The amount of superoxide anion wascalculated using extinction coefficient of 4 mM�1 cm�1 andexpressed as nmol g�1 fresh wt. All the experiments were carriedout in sealed tube under N2 atmosphere to minimize oxidation andgeneration of ROS.

4.4. Superoxide dismutase (SOD) assay

The superoxide dismutase (SOD, EC 1.15.1.1) activity was assayedas earlier [7]. About 200 mg fresh leaf samples were taken fromcontrol and drought stressed tomato plants and homogenized in5 ml of 100 mM potassiumephosphate buffer (pH 7.8), containing0.1 mM EDTA, 0.1% (v/v) Triton X-100 and 2% (w/v) of solublepolyvinyl pyrrolidone (PVP) using prechilled mortar and pestle.Extracts were centrifuged at 22,000 � g for 10 min at 4 �C and SODactivity was assayed in the supernatant. The assay mixture con-tained 50 mM sodium carbonateebicarbonate buffer (pH 9.8),containing 0.1 mM EDTA, 0.6 mM epinephrine and enzyme ina total volume of 3 ml. Epinephrine was the last component to beadded. The adrenochrome formation during the next 5 min wasrecorded at 470 nm in a UVeVis spectrophotometer (ELICO, SL-159,India). One unit of SOD activity is defined as the amount of enzymerequired to cause 50% inhibition of epinephrine oxidation under theexperimental conditions.

4.5. Catalase (CAT) assay

Catalase (EC 1.11.1.6) was assayed as earlier [7]. About 200 mgfresh leaf samples were homogenized in 5 ml of 50 mM TriseNaOHbuffer (pH 8.0) containing 0.5 mM EDTA, 2% (w/v) PVP and 0.5% (v/v) Triton X-100 using a chilled mortar and pestle. The homogenatewas centrifuged at 22,000 � g for 15 min at 4 �C and supernatantwas used for enzyme assay. Assay mixture in a total volume of1.5 ml contained l000 ml of 100 mM KH2PO4 buffer (pH 7.0), 400 mlof 200 mM H2O2 and 100 ml enzyme. Decrease in H2O2 was moni-tored at 240 nm (extinction coefficient of 0.036 mM�1 cm�1).Enzyme specific activity is expressed as mmol of H2O2 oxidizedmg�1 (protein) min�1.

4.6. Ascorbate peroxidase (APX) assay

Ascorbate peroxidase (APX, EC1.11.1.11) was assayed as earlier [7].About 200 mg of fresh leaf samples were homogenized in 5 ml of50 mM potassiumephosphate buffer (pH 7.8) containing 1% PVP,1 mM EDTA and 1 mM ascorbic acid (added just before use) ina chilled mortar and pestle. The homogenate was centrifuged at22,000 � g at 4 �C for 15 min and the supernatant was used forenzyme assay. Reaction mixture in a total volume of 3 ml contained50 mM potassiumephosphate buffer (pH 7.0) containing 0.1 mMEDTA and 0.5 mM ascorbic acid, 0.1 mM H2O2 and 0.1 ml enzymeextract.H2O2was the last component tobeaddedand the absorbancewas recorded at 290 nm (extinction coefficient 2.8 mM�1 cm�1).Enzyme specific activity is expressed as mmol ascorbate oxidizedmg�1 (protein) min�1.

4.7. Glutathione reductase (GR) assay

Glutathione reductase (GR, EC 1.6.4.2) was assayed as before [7]with modifications. About 200 mg of fresh leaf tissues werehomogenized in 5ml of 0.1M potassiumephosphate buffer (pH 7.0)using chilled mortar and pestle. The homogenate was centrifugedat 22,000 � g for 10 min at 4 �C and the supernatant obtained wasused for determination of enzyme activity. The reaction mixturecontained 0.25 mM GSSG, 0.125 mM NADPH, 50 mM tricine (pH7.8), 0.5 mM EDTA and 50 ml of extract in a final volume of 2 ml. Thedecrease in absorbance due to NADPH oxidation was recorded at340 nm (extinction coefficient of 6.22 mM�1 cm�1) and expressedin terms of nmol NADPH oxidized mg�1 (protein) min�1.

4.8. Guaiacol peroxidase (POD) assay

The activity of guaiacol peroxidase (POD, EC1.11.1.7) was assayedspectrophotometrically as before [7]. POD was extracted byhomogenizing about 200 mg of leaf samples in 5 ml of 60 mMphosphate buffer (pH 6.0) using a chilled mortar and pestle at 4 �C.The homogenates were centrifuged at 22,000 � g for 10 min andsupernatant were used for enzyme assay. Assay mixture in a finalvolume of 2 ml contained 50 ml enzyme, 200 ml guaiacol and 50 mlH2O2 in 1.7 ml of buffer. The increase in absorbance was measuredat 470 nm (extinction coefficient 26.6 mM�1 cm�1) using an ElicoSL-159 (India) spectrophotometer. Enzyme specific activity isexpressed as mmol H2O2 reduced mg�1 (protein) min�1.

4.9. Estimation of proline

Proline was estimated in tomato leaves according to Shah andDubey [9]. A 200 mg fresh leaf samples were homogenized in 5 mlof 3% aqueous sulphosalycylic acid and centrifuged at 22,000 � gfor 5 min. To 2 ml of supernatant 2 ml of acid ninhydrin was added.Further, 2 ml of glacial acetic acid was added to the mixture andboiled in a water bath at 100 �C for 1 h. The mixture was furtherextracted with 4 ml of toluene by mixing the two thoroughly ina test tube with vigorous stirring. Absorbance of chromophore wasread at 520 nm against toluene as blank in an Elico SL-159 spec-trophotometer (India). L-Proline (Merck) was used for the prepa-ration of the standard curve. The amount of proline in the sampleswas calculated in mg proline g�1 fresh weight.

4.10. Protein determination

In all the enzyme preparations protein was determined by theLowry’s method [40] using bovine serum albumin (BSA, Himedia)as standard.

4.11. Statistical analysis

The data were analyzed by a simple variance analysis (ANOVA)and significant difference were compared by t-test.

Acknowledgments

The work is a part of Network Project on Transgenic Crops,funded by ICAR, New Delhi. The valuable inputs while preparationof the manuscript by Dr. P.S. Naik, Director, IIVR is gratefullyacknowledged. We thank Prof. K.C. Bansal, NRCPB, Pusa Campus,New Delhi for providing the ZAT12 gene construct. Authors arethankful to Mr. V. Agrawal (U.K.) for language editing.

There is no conflict of interest and the contributions of all theauthors are equal.

A. Chandra Rai et al. / Plant Physiology and Biochemistry 61 (2012) 108e114114

References

[1] K. Shah, S. Nahakpam, Heat stress and cadmium toxicity in higher plants e anoverview, in: A. Hemantranjan (Ed.), Advances in Plant Physiology, vol. 12,Scientific Publishers, Jodhpur, India, 2011, pp. 243e280.

[2] D. Fleury, S. Jefferies, H. Kuchel, P. Langridge, Genetic and genomic tools toimprove drought tolerance in wheat, J. Exp. Bot. 61 (2010) 3211e3222.

[3] N. Isendahl, G. Schmidt, Drought in the Mediterranean-WWF Policy Proposals,A WWF Report, Madrid (2006).

[4] H. Nayyar, D. Gupta, Differential sensitivity of C3 and C4 plants to water deficitstress: association with oxidative stress and antioxidants, Environ. Exp. Bot.58 (2006) 106e113.

[5] A. Polle, Dissecting the superoxide dismutase-ascorbate glutathione pathwayin chloroplasts by metabolic modeling. Computer simulations as a steptowards flux analysis, Plant Physiol. 126 (2001) 445e462.

[6] K. Shah, R.G. Kumar, S. Verma, R.S. Dubey, Effect of cadmium on lipidperoxidation, superoxide anion generation and activities of antioxidantenzymes in growing rice seedlings, Plant Sci. 161 (2001) 1135e1144.

[7] S. Nahakpam, K. Shah, Expression of key antioxidant enzymes undercombined effect of heat and cadmium toxicity in growing rice seedlings, PlantGrowth Regul. 63 (2011) 23e35.

[8] C.J. Howarth, Genetic improvements of tolerance to high temperature, in:M.Ashraf, P.J.C.Harris (Eds.), Abiotic Stresses: PlantResistanceThroughBreedingand Molecular Approaches, Howarth Press Inc., New York, 2005, pp. 277e280.

[9] K. Shah, R.S. Dubey, Effect of cadmium on proline accumulation and RNaseactivity in rice Seedlings: role of proline as a possible enzyme protectant, Biol.Plant 40 (1997/98) 121e130.

[10] S.S. Gill, N. Tuteja, Reactive oxygen species and antioxidant machineryin abiotic stress tolerance in crop plants, Plant Physiol. Biochem. 48 (2010)909e930.

[11] A. Mueller, S.D. Tanksley, J.J. Giovannoni, J. Van Eck, S. Stack, D. Choi, B.D. Kim,M. Chen, et al., The tomato sequencing project, the first corner stone ofthe international Solanaceae project (SOL), Comp. Funct. Genomics 6 (2005)153e158.

[12] D. Ruma, M.S. Dhaliwal, A. Kaur, S.S. Gosal, Transformation of tomato usingbiolistic gun for transient expression of the b- glucuronidase gene,Ind. J. Biotechnol. 8 (2009) 363e369.

[13] A.C. Rai, M. Singh, K. Shah, Environmental stresses and transgenics: role of ZFP(ZAT) gene in multiple stress tolerance in plants, in: A. Hemantranjan (Ed.),Advances in Plant Physiology, vol. 13, Scientific Publishers, Jodhpur, India,2012, pp. 197e232.

[14] S. Davletova, K. Schlauch, J. Coutu, R. Mittler, The zinc-finger protein Zat12plays a central role in reactive oxygen and abiotic stress signaling inArabidopsis, Plant Physiol. 139 (2005) 847e856.

[15] A.L. Garcia, R. Madrid, N. Nicolas, V. Martinez, J.A. Franco, Moderating waterstress in tomato (Lycopersicon esculentum Mill.) plants by application ofspecific nitrogen doses, J. Hort. Sci. Biotechnol. 82 (2007) 664e670.

[16] M. Menconi, C.L.M. Sgherri, C. Pinzino, F. Navari-Izzo, Activated oxygenproduction and detoxification in wheat plants subjected to a water deficitprogram, J. Exp. Bot. 46 (1995) 1123e1130.

[17] C.B. Ahmed, B.B. Rouina, S. Sensoy, M. Boukhris, F.B. Abdallah, Changes in gasexchange, proline accumulation and antioxidative enzyme activities in threeolive cultivars under contrasting water availability regimes, Environ. Exp. Bot.67 (2009) 345e352.

[18] Y. Tateishi, T. Nakagawa, M. Esaka, Osmotolerance and growth stimulation oftransgenic tobacco cells accumulating free proline by silencing prolinedehydrogenase expression with double-stranded RNA interference technique,Physiol. Plant 125 (2005) 224e234.

[19] E. Esfandiari, M.R. Shakiba, S.A. Mahboob, H. Alyari, S. Shahabivand, The effectof water stress on the antioxidant content, protective enzyme activities,proline content and lipid peroxidation in wheat seedling, Pak. J. Biol. Sci. 11(2008) 1916e1922.

[20] K. Shah, S. Nahakpam, Heat exposure alters the expression of SOD, POD,APX and CAT isoenzymes and mitigates low cadmium toxicity in seedlings

of sensitive and tolerant rice cultivars, Plant Physiol. Biochem. 57 (2012)106e113.

[21] K. Asada, Production and action of active oxygen species in photosynthetictissue, in: C.H. Foyer, P.M. Mullineaux (Eds.), In Causes of PhotooxidativeStress and Amelioration of Defense Systems in Plants, CRC Press, Boca Raton,1994, pp. 77e104.

[22] H.P. Misra, I. Fridovich, The role of superoxide anion in the autooxidation ofepinephrine and a simple assay for superoxide dismutase, J. Biol. Chem. 247(1972) 3170e3175.

[23] M. Faize, L. Burgos, L. Faize, A. Piqueras, E. Nicolas, G. Barba-Espin,M.J. Clemente-Moreno, R. Alcobendas, T. Artlip, J.A. Hernandez, Involvementof cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase forimproved tolerance against drought stress, J. Exp. Bot. 62 (2011) 2599e2613.

[24] C.C. Lin, C.H. Kao, Effect of NaCl stress on H2O2 metabolism in rice leaves, PlantGrowth Regul. 30 (2000) 151e155.

[25] E.A. MacRae, I.B. Ferguson, Changes in catalase activity and hydrogen peroxideconcentration in plants in response to low temperature, Physiol. Plant 65(1985) 51e56.

[26] R. Mittler, B.A. Zilinskas, Regulation of pea cytosolic ascorbate peroxidase andother antioxidant enzymes during the progression of drought stress andfollowing recovery from drought, Plant J. 5 (1994) 397e405.

[27] M.H.C. Carvalho, Drought stress and reactive oxygen species, Plant Signal.Behav. 3 (2008) 156e165.

[28] Z. Guo, W. Ou, S. Lu, Q. Zhong, Differential responses of antioxidative systemto chilling and drought in four rice cultivars differing in sensitivity, PlantPhysiol. Biochem. 44 (2006) 828e836.

[29] B.D. McKersie, S.R. Bowley, K.S. Jones, Winter survival of transgenicalfalfa overexpressing superoxide dismutase, Plant Physiol. 119 (1999)839e848.

[30] J. Zhang, M.B. Kirkham, Antioxidant responses to drought in sunflower andsorghum seedlings, New Phytol. 132 (1996) 361e373.

[31] S. Shigeoka, T. Ishikawa, M. Tamoi, Y. Miyagawa, T. Takeda, Y. Yabuta,K. Yoshimura, Regulation and function of ascorbate peroxidase isoenzymes,J. Exp. Bot. 53 (2002) 1305e1319.

[32] R.G. Alscher, Biosynthesis and antioxidant properties of glutathione in plants,Physiol. Plant 77 (1989) 457e464.

[33] R. Baisak, D. Rana, P.B.B. Acharya, M. Kar, Alteration in the activities of activeoxygen scavenging enzymes of wheat leaves subjected to water stress, PlantCell Physiol. 35 (1994) 489e495.

[34] G. Szalai, T. Kellos, G. Galiba, G. Kocsy, Glutathione as an antioxidant andregulatory molecule in plants under abiotic stress conditions, J. Plant GrowthRegul. 28 (2009) 66e80.

[35] J.P. Riechheld, M. Khafif, C. Riondet, M. Droux, G. Bonnard, Y. Meyer,Inactivation of thioredoxin reductases reveals a complex interplay betweenthioredoxin and glutathione pathways in Arabidopsis development, Plant Cell19 (2007) 1851e1865.

[36] P.D. Hare, W.A. Cress, Metabolic implications of stress-induced prolineaccumulation in plants, Plant Growth Regul. 21 (1997) 79e102.

[37] T. Nanjo, M. Kobayashi, Y. Yoshiba, Y. Sanada, K. Wada, H. Tsukaya,Y. Kakubari, K. Yanaguchi-Shinozaki, K. Shinozaki, Biological functions ofproline in morphogenesis and osmotolerance revealed in antisense transgenicArabidopsis thaliana, Plant J. 18 (1999) 185e193.

[38] K. Shah, C. Penel, J. Gagnon, C. Dunand, Purification and identification ofa Ca2þ-pectate binding peroxidase from Arabidopsis leaves, Phytochemistry 65(2004) 307e312.

[39] J.A. O’Brien, A. Daudi, P. Finch, V.S. Butt, J.P. Whitelegge, P. Souda,F.M. Ausubel, G.P. Bolwell, A peroxidase-dependent apoplastic oxidative burstin cultured Arabidopsis cells functions in MAMP-elicited defense, PlantPhysiol. 158 (2012) 2013e2027.

[40] O.H. Lowry, R.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement withfolin- phenol reagent, J. Biol. Chem. 193 (1951) 265e275.

[41] A.C. Rai, M. Singh, K. Shah, Engineering drought tolerant tomato plants over-expressing BcZAT12 gene encoding a C2H2 zinc finger transcription factor,Phytochemistry (2012). in press.