the effects of brassinosteroid on the induction of...

IntroductionDrought stress is defined as a condition in which

water available to plants is so low that it isunfavourable for the growth of a plant species (Zhu2001 Egert amp Tevini 2002) Plants will respond toconditions of drought stress through a number ofphysiological and developmental changes (Inze amp

Montagu 2000) Under stressful conditions the stressfactors or the toxic molecules derived from the stressattack the most sensitive molecules (primary targets)in cells to impair their function (Ingram amp Bartels1996 Inze amp Montagu 2000) The damaged targetsrecover either by repair or replacement via de novobiosynthesis When the stress is too intense and

417

Research Article

Turk J Bot33 (2009) 417-428copy TUumlBİTAKdoi103906bot-0806-12

The effects of brassinosteroid on the induction of biochemicalchanges in Lycopersicon esculentum under drought stress

Mehri BEHNAMNIA12 Khosrow Manouchehri KALANTARI2 Jalal ZIAIE3

1Department of Biology Faculty of Science Basic Science University of Damghan Damghan I R of IRAN2Department of Biology Faculty of Science Shahid Bahonar University Kerman I R of IRAN

3Agri-Jahad Semnan Semnan I R of IRAN

Received 30062008Accepted 04112009

Abstract Drought stress is considered a restricting factor for plant products therefore many compounds were appliedto minimise the harmful effects of stress One type of these compounds that have antioxidative characteristics isbrassinosteroids In this experiment when 4 fully expanded leaves of tomato plants appeared 24-epibrassinolide (24-EBL)was sprayed onto the leaves at 001 and 1 μM concentrations for 3 days with a 1-day interval Three levels of drought stress(0 3 and 5 days withholding water) were applied Thereafter the effects of brassinosteroids and water stress wereinvestigated on some biochemical and antioxidative parameters of tomato plants Lipid peroxidation peroxide hydrogenand proline content increased in plants subjected to drought stress Reduction in protein content in drought conditionsshowed that drought stress affected protein synthesis and degradation Decline in the activity of antioxidant enzymes(POD and APX) and increases in the SOD GR and CAT activities were observed in drought stressed plants Reductionin peroxidation of lipids and H2O2 content in the plants treated with both 24-EBL and drought stress was observed Basedon our results it seems that brassinosteroids considerably alleviated oxidative damage that occurred under drought stressIncrease in activity of antioxidant enzymes (POD CAT APX GR and SOD) and change in isoenzymes pattern withhigher intensity as well as increases in proline and protein content in drought stressed plants treated with BR will probablyshow the role of brassinosteroids in increased tolerance of plants to water stress Therefore 24-epibrassinolide may havea role in the mitigation of damage caused by water stress

Key words Drought stress brassinosteroid antioxidative tomato proline lipid peroxidation

E-mail mbehnamniayahoocom

severely damages target molecules catastrophiccascades of events set in leading to cell death (Inze ampMontagu 2000) Cells could be protected either by theendogenous molecular systems or exogenous appliedcompounds that mitigate the stress (Ingram amp Bartels1996) Capacity of plants to detoxify reactive oxygenspecies has been related to the stress tolerance ofplants (Egert amp Tevini 2002 Apel amp Hirt 2004)

Oxidative stress is a key underlying component ofmost abiotic stresses (Mittler 2002 Apel amp Hirt 2004Yin et al 2008) and a major limiting factor to plantgrowth in the field (Scandalios 2002 Blokhina et al2003 Mittler 2006) Production of reactive oxygenspecies (ROS) and other radicals increasesdramatically during abiotic stress conditions andoxidative stress occurs when the rate of ROSproduction outstrips the capacity of antioxidantsystems to detoxify them (McCord 2000 Mittler2002 Mittler et al 2004) The result is oxidativedamage to key biomolecules such as proteins lipidsand DNA leading to cellular dysfunction andultimately cell death (Wagner et al 2004 Halliwell2006 Baxter et al 2007) To avoid oxidative damageplants invoke a molecular response that allows themto cope with and adapt to the oxidative stresssituation Many stress-related genes have beenidentified and the list of antioxidant enzymeshormones and metabolites present in plantscontinues to be extended (Nakashita et al 2003 Caoet al 2005) One type of these compounds that haveantioxidative characteristics is brassinosteroids (BRs)(Bishop amp Koncz 2002 Haubrick amp Assmann 2006)

BRs are common plant-produced compounds thatcan function as growth regulators (Khripach et al2000 Davis 2005 Bishopp et al 2006) In addition ithas been suggested that BRs could be included in thecategory of phytohormones (Clouse amp Feldmann1999 Haubrick amp Assmann 2006) Exogenousapplication of BRs may influence a range of diverseprocesses of growth and development in plants (Caoet al 2005 Clouse amp Sasse 1998 Yu et al 2004Montoya et al 2005) In addition it is becoming clearthat BRs interact both negatively and positively withother major signalling pathways including thoseregulated by auxin and ethylene (Amzallage ampVaiseman 2006 Haubrick et al 2006) It is clear thatBRs provide protection against a number of abiotic

stresses (Sasse 1999 Rao amp Vardhini 2002 Krishna2003 Nemhauser 2004) Treatment with BRsenhanced the growth of wheat (Sairam 1994) Frenchbean (Upreti amp Murti 2004) and sugar beet plants(Schilling 1991) under drought stress Application ofBRs improved tolerance against salt in rice (Anuradhaamp Rao 2001 2003 Oumlzdemir et al 2004) groundnut(Vardhini amp Rao 1997) wheat (Sairam et al 2005)and chickpea (Ali et al 2007) It was reported that BLinduced thermotolerance in tomato (Dhaubhadel etal 1999 Mazorra et al 2002 Ogweno et al 2008)rice (Wang amp Zang 1993) and brome grass (Wilen etal 1995) Moreover BRs increased tolerance againsthigh temperatures in Brassica napus (Singh amp Shono2005) and brome grass (Wilen et al 1995) Onemechanism that may be involved in the resistance tomany types of stress is the increased activity of theantioxidant pathway Several studies have shown thatBRs alter the antioxidant capacity of plants understress conditions (Kamuro amp Takatsuto 1999Yardanova 2004 Yin et al 2008) The present studywas an attempt to carry out investigations of the effectof brassinolide on the water stress responses of tomatoplants with the aim to elucidate possible mechanismsthat might be involved in the BR-promotedantioxidant responses to drought

Abbreviations BRndashBrassinosteroid MDAndashMalondialdehyde TBAndashThiobarbituric Acid TCAndashTrichloroacetic Acid EBL-EpibrassinolideHBLndashHomobrassinolide CATndashCatalase PODndashPeroxidase APXndashAscorbate peroxidase ROSndashReactive Oxygen Species SOD-Superoxide dismutaseH2O2- peroxide hydrogen NBT- ρ-nitro bluetetrazolium chloride EDTA-Ethylendiaminetetraacetic acid GR- Glutathione reductase

Materials and methods24-Epibrassinolide (EBL C28H48O6 MW = 480)

was purchased from Sigma (USA) Tomato(Lycopersicon esculentum Mill var Tomba (BB204))seeds were used in this study Seeds were sown inplastic pots containing sand loam and peat (211)When 4 fully expanded leaves appeared (the tomatoplants were 1 month old) the tomato was left to growin 2618 degC and 168 h (lightdark) conditions in agrowth chamber Hoaglandlsquos nutrient solutions were

The effects of brassinosteroid on the induction of biochemical changes in Lycopersicon esculentum under drought stress

418

used every 7 days and the pH was maintained at about67 (Meidner 1984) 24-EBL was dissolved in distilledwater and ethanol Tween-20 (001) was used assurfactant Preliminary screening was performed forvarious concentration of 24-epibrassinolide to obtainthe optimum responses and the concentrations of 001and 1 μM 24-EBL were selected 24-EBL was sprayedonto the leaves at 001 and 1 μM concentration for 3days on alternate days Three levels of water stress(03 and 5 days withholding water) were appliedLight intensity was 6000 Lux at the plant level Fourreplicates were assigned for each treatment Aftertreatment the third leaves of plants were harvestedand samples were either rapidly dried in an oven at 80degC to constant weight which were used fordetermination of dry weight and further analyses orwere frozen in liquid nitrogen and stored at ndash80 degCfor biochemical analysis

Biochemical assaysThe level of lipid peroxidation in plant tissues was

measured by determination of MDA (Heath amp Packer1969) and others aldehydesrsquo (Meirs et al 1992)breakdown products of lipid peroxidation MDAcontent was determined with a thiobarbituric acid(TBA) reaction Briefly a 02 g tissue sample washomogenised in 5 mL of 01 TCA The homogenatewas centrifuged at 10000 timesg for 5 min To a 1 mLaliquot of the supernatant was added 4 mL of 20TCA containing 05 TBA The mixture was heatedat 95 degC for 15 min and cooled immediately Theabsorbance was measured at 532 nm The value forthe non-specific absorption at 600 nm was subtractedThe level of lipid peroxidation was expressed as μmolof MDA formed using an extinction coefficient of 155mM-1 cm-1 and of others aldehydes formed theextinction coefficient was 0457 times 105 M-1 cm-1

Proline was extracted and its concentrationdetermined as described by Bates et al (1973) Leaftissues were homogenised with 3 sulfosalicylic acidand the homogenate was centrifuged at 3000 timesg for20 min The supernatant was treated with acetic acidand acid ninhydrin boiled for 1 h and thenabsorbance at 520 nm was determined Proline(Sigma) was used for the standard curve

Soluble proteins were determined as described byBradford (1976) using BSA (Merck) as standard

The content of H2O2 was determined according toAlexieva et al (2001) Leaf tissue (500 mg) washomogenised in an ice bath with 5 cm3 of cold 01(mv) trichloroacetic acid (TCA) The homogenatewas centrifuged (12000 timesg 15 min 4 degC) and 05 cm3

of the supernatant was added to 05 cm3 of 100 mMpotassium phosphate buffer (pH 70) and 1 cm3 of 1M KI The absorbance was read at 390 nm

Enzyme AssaysFrozen leaf samples (05 g) were used for enzyme

extraction Samples were ground in 2 mL of 50 mMphosphate buffer (pH 72) using a pre-chilled mortarand pestle Phosphate buffer contained 1 mM EDTA1 mM PMSF and 1 PVP-40 Then the extract wascentrifuged at 4 degC at 17000 timesg for 10 min

The activity of ascorbate peroxidase (APX EC111111) enzyme was measured using the method ofNakano and Asada (1981) The reaction mixtureconsisted of 50 mM potassium phosphate buffer (pH70) containing 05 mM ascorbic acid 015 mM H2O201 mM EDTA and 50 μL of enzyme extract(supernatant) Oxidation of ascorbic acid wasconsidered a decrease in absorbance at 290 nm thatwas followed 2 min after starting the reaction Oneunit of APX oxidises 1 mM ascorbic acid in 1 min at25 degC

Catalase (CAT EC 11116) activity was assayedwith spectrophotometry by monitoring the decreasein absorbance of H2O2 at 240 nm using the method ofDhindsa et al (1981) The assay solution contained 50mM potassium phosphate buffer (pH 70) and 15 mMH2O2 The reaction was started by the addition of 100μL of enzyme extract to the reaction mixture and thechange in absorbance was followed 1 min afterstarting of the reaction Unit of activity was taken asthe amount of enzyme that decomposes 1 mM ofH2O2 in 1 min

Peroxidase (POD EC 11117) activity wasdetermined using the guaiacol test (Plewa et al 1991)The tetraguaiacol formed in the reaction has amaximum absorption at 470 nm and thus the reactioncan be readily followed photometrically The enzymewas assayed in a solution that contained 50 mM

M BEHNAMNIA K M KALANTARI J ZIAIE

419

phosphate buffer (pH 70) 03 H2O2 and 1guaiacol The reaction was started by the addition of20 μL of the enzyme extract at 25 degC and was followed3 min after starting the reaction The enzyme unit wascalculated for the formation of 1 mM tetraguaiacol for1 min

A photochemical method published byGiannopolitis and Reis (1977) was used to determinesuperoxide dismutase (SOD EC 11511) activity Thereaction solution (3 mL) contained 50 μM NBT 13μM riboflavin 13 mM methionine 75 nm EDTA 50mM phosphate buffer (pH 78) and 20-50 μL ofenzyme extract The test tubes containing the reactionsolution were irradiated under light (15 fluorescentlamps) at 78 μmol m-2 s-1 for 15 min The absorbanceof the irradiated solution was read at 560 nm using aspectrophotometer (Cary 50) One unit of SODactivity was defined as the amount of enzyme thatinhibited 50 ρ-nitro blue tetrazolium chloride(NBT) photoreduction

Glutathione reductase (GR EC 1642) activitywas measured according to Foyer and Halliwell(1976) which depends on the rate of decrease in theabsorbance of oxidised glutathione (GSSG) at 340 nmThe reaction mixture contained 25 mM Na-phosphate buffer (pH 78) 5 mM GSSG and 12 mMNADPHNa4 The reaction was carried out for 3 minand activity of GR was calculated from the reducedGSSG concentration by using the extinctioncoefficient 62 mM-1 cm-1 One enzyme unit wasdefined as μmol mL-1 oxidised GSSG per min

Native PAGE and activity stainNative polyacrylamide gel electrophoresis (PAGE)

was performed at 4 degC 180 V following Laemmli(1970) For APX the enzyme solutions were subjectedto native PAGE with 12 polyacrylamide gel forPOD with 10 and for CAT with 7 SDS wasomitted from the PAGE Activity stain for eachenzyme was carried out as follows APX activity wasdetected by the procedure described by Mittler andZilinskas (1993) Gel equilibrated with 50 mMsodium phosphate buffer (pH 70) containing 2 mMascorbate for 30 min was incubated in a solutioncomposed of the same buffer 4 mM ascorbate and 2mM H2O2 for 20 min Then the gel was washed in thebuffer for 1 min and submerged in a solution of thefollowing buffer containing 28 mM TEMED and 245

mM NBT for 10-20 min with gentle agitation in thepresence of light For POD gel was incubated in 25mM potassium phosphate buffer (pH 70) and thenthe gel was submerged again in the same buffer (freshamount) containing 18 mM guaiacol and 25 mMH2O2 until the POD activity-containing band wasvisualised carefully (Fielding and Hall 1978) ForCAT gel was incubated in 327 mM H2O2 solution for25 min and then the gel was washed in distilled waterand submerged in a solution composed of potassiumferricyanide 1 and ferric chloride 1 for 4 min(Woodbury et al 1971)

Statistical analysisAll experiments were performed with 4 replicates

using a completely randomised design Data werestatistically analysed by one-way analysis of varianceusing SPSS (version 11) and the means were separatedby Duncanrsquos multiple range test at 005 probabilitylevel

Results and discussionThe content of proline progressively increased in

plants as the drought levels increased (Figure 1) Inthose plants that were either under drought stress or24-EBL treatments increases in the content of prolinewere observed It has been reported that BRtreatments induced expressing of biosynthetic genes


420

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e e e

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a

Figure 1 Effects of BR and drought stress on proline content ofLycopersicon esculentum Plants were grown for 1month under controlled conditions and were dividedinto 3 groups Two groups were pretreated with BR andthe other group was just sprayed with distilled waterAll 3 groups were exposed to water stress for 3 and 5days The control group was irrigated daily Values arethe means of 4 replicates and letters show significantdifferences among the means at P lt 005 according toDuncanrsquos test

of proline (Oumlzdemir et al 2004) There are alsoreports that show that application of BRs (28-HBLand 24-EBL) increased proline content in sorghumplants exposed to osmotic stress (Vardhini amp Rao2003) Proline as a cytosolic osmoticum and as ascavenger of OH radical can interact with cellularmacromolecules such as enzymes DNA andmembranes and stabilise the structure and functionof such macromolecules (Anjum et al 2000 Matysiket al 2002 Kavir Kishor et al 2005) In additionamong various compatible solutes proline is the onlymolecule that has been shown to protect plantsagainst singlet oxygen and free radical induceddamage resulting from stress (Alia et al 1997) Ourdata showed that proline was accumulated in BR-treated plants under stress conditions Therefore arole of BR in the accumulation of proline as animportant component of protective reactions intomato plants in response to drought stress is possible

Formation of MDA and others aldehydes in plantsexposed to water stress is a reliable indicator of freeradical formation in the tissue and is currently usedas an indicator of lipid peroxidation (Meirs et al

1992 Borsanio et al 2001) Considering that droughtstress results in increases in reactive oxygen species(Apel amp Hirt 2004 Gong et al 2006) peroxidationof lipid membranes is both a reflection and measureof stress-induced damage at the cellular level (Meirs etal 1992 Borsanio et al 2001) In the present studythe content of MDA other aldehydes and H2O2increased significantly as drought progressed in plants(Figures 2-4) It was previously found that the MDAcontent increased in 3 genotypes of wheat underdrought stress (Sgherri et al 2000) It is affirmed thatunder water stress the content of MDA and H2O2 isdifferent among different cultivars of bean bananaand rice In drought-tolerant cultivars since there ishigh antioxidative potential they are able to scavengeH2O2 radicals and with decreasing H2O2 content lowerMDA content is produced compared to sensitive-tolerance cultivars (Li amp Van Staden 1998 Sharma ampShanker 2005 Tuumlrkan et al 2005) Furthermore inLilium longiflorum plants under short-term heatstress H2O2 and MDA contents slightly varied at 37and 42 degC while increasing significantly at 47 degC (Yinet al 2008) In our study BR-pretreated plants that


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)


c cc

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aab ab

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(nM

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)


ef fg

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Figures 2-4 Effects of BR and drought stress on MDA other aldehydes and H2O2 content of L esculentum Plants were grown for 1month under controlled conditions and were divided into 3 groups Two groups were pretreated with BR and the othergroup was just sprayed with distilled water All 3 groups were exposed to water stress for 3 and 5 days The control groupwas irrigated daily Values are the means of 4 replicates and the letters show significant differences among the means at Plt 005 according to Duncanrsquos test

2

3 4

were thereafter put under drought stress had muchless H2O2 MDA and others aldehydesrsquo content thandrought stressed plants (Figures 2 and 3) It isassumed that BRs act as secondary messengers for theinduction of antioxidant defences in stressed plants(Khripach et al 2000) thus based on our results inMDA measurement it is very possible that 24-EBLeffectively scavenged ROS by increasing the activityof antioxidant enzyme systems

A decrease in protein content is a commonphenomenon in drought stress (Zhu 2001) Thereason for this is that the amino acid of proteins willreact with active radical and will be degraded (Wagneret al 2004) In this study decreases in protein contentin water stressed plants were observed 24-EBLapplication increased the protein content both innormal and stressed plants (Figure 5) BRs participatein the processes of gene expression transcription andtranslation in normal and stressed plants (Sasse 1999Mazorra et al 2002) It has been observed that thetolerance to high temperature in tomato leaves(Dhaubhadel et al 1999 Ogweno et al 2008) andbrome grass (Kulaeva et al 1991) is due to applicationof BRs which is associated with induction of de novopolypeptide synthesis The promotion growth inseedlings by BRs under saline conditions was relatedto enhanced levels of nucleic acids and solubleproteins (Anuradha amp Rao 2001 2003 Shahbaz et al2008) In addition Sairam (1994) reported the

enhancement of proteins in wheat plants byhomobrassinolide under moisture stress conditionsIt has been reported that osmotic stress resulted inconsiderable reductions in the protein content in theseedlings of both susceptible and tolerant varieties ofsorghum plants but treatment with brassinosteroidsresulted in not only restoring the protein levels butalso further improvement (Vardhini amp Rao 2003) Inthis study with pretreatment with BR when plantswere under severe drought protein content was muchlower than that in mild stress Possibly inductionoxidative stress is responsible because proteindegradation may occur under severe drought stressA number of reports have shown that cells exhibitincreased rates of proteolysis following exposure tooxidative-inducing agents (Inze amp Montagu 2000Jung 2003)

In the present study drought stress decreased theperoxidase activity in 5-day drought stressed plants(Figure 6) 24-EBL application positively influencedPOD activity in control and stressed plants Thehighest activity was observed in control and 5-daydrought stressed plants The activity of enzyme was2-fold higher in those plants treated with 1 μM BRwhen compared with BR-free plants (Figure 6) In 5-day drought stress conditions the activity of catalasesignificantly increased (Figure 7) 24-EBLpretreatment increased CAT activity in droughtstressed plants In the severe drought stressed plantsthe activity was much higher than that in the controlunstressed plants (Figure 7) Drought stress resultedin a decrease in ascorbate peroxidase (APX) activityIn those plants under 5-day drought APX activity was33 less than that in unstressed plants (Figure 8) 24-EBL application caused an increase in APX activity inthe control and 5-day drought stressed plants Thehighest activity was observed under condition of 1 μM24-EBL and 5-day drought and that was about 4 timesmore than that in BR-free stressed plants (Figure 8)Drought stress significantly increased the activity ofSOD Exogenous application of EBL in droughtstressed plants enhanced the activity of this enzymeat both levels of 24-EBL concentration (Figure 9)Drought treatment induced GR activity on both days3 (30) and 5 (65) in tomato plants (Figure 10)Moreover the induced level of GR activity in 24-EBLtreated groups was higher on both days 3 and 5 daysafter withholding water


422

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g F

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bcab

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de cd


0 μM BR

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Figure 5 Effects of BR and drought stress on protein content ofL esculentum Plants were grown for 1 month undercontrolled conditions and were divided into 3 groupsTwo groups were pretreated with BR and the othergroup was just sprayed with distilled water All 3 groupswere exposed to water stress for 3 and 5 days Thecontrol group was irrigated daily Values are the meansof 4 replicates and the letters show significantdifferences among the means at P lt 005 according toDuncanrsquos test

In part of the native polyacrylamide gelelectrophoresis (PAGE) analysis 4 isoenzymes weredetected for peroxidase enzyme especially in BR-treatment and drought stress conditions (Figure 11)In addition one isoenzyme was detected for CATenzyme Especially in BR-treated drought stressedplants CAT isoenzyme showed higher intensity(Figure 12) Native polyacrylamide gel electrophoresis

(PAGE) analysis on ascorbate peroxidase enzymeshowed 4 isoenzymes for this enzyme In BR-treateddrought stressed plants these isoenzymes weremarkedly detected (Figure 13)

The increase in peroxidase activity that wasobserved in our study could reflect a similar processof oxidative stress with the implication of peroxidaseactivity as part of the antioxidant response against


423

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Figures 6 7 Effects of BR and drought stress on POD and CAT activity of Lesculentum Plants were grown for 1 month under controlledconditions and were divided into 3 groups Two groups were pretreated with BR and the other group was just sprayedwith distilled water All 3 groups were exposed to water stress for 3 and 5 days The control group was irrigated dailyValues are the means of 4 replicates and the letters show significant differences among the means at P lt 005 according toDuncanrsquos test

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Figures 8-10 Effects of BR and drought stress on APX SOD and GR activity of L esculentum Plants were grown for 1 month undercontrolled conditions and were divided into 3 groups Two groups were pretreated with BR and the other group was justsprayed with distilled water All 3 groups were exposed to water stress for 3 and 5 days The control group was irrigateddaily Values are the means of 4 replicates and the letters show significant differences among the means at P lt 005according to Duncanrsquos test

6 7

8

9 10

H2O2 Ascorbate peroxidases (APXs) are the mostimportant H2O2 scavengers operating in both thecytosol and chloroplasts (Yardanova et al 2004) Inthe present work drought stress decreased the activityof POD and APX enzymes under 5-day drought stressbut CAT GR and SOD activities increased (Figures7 9 10) The reason for this decrease could be thedecrease in the amount instead of enzyme activitybecause in this condition protein content decline alsocan be attributed to an increase in CAT activity Theseresults were accompanied by increases in MDA andproline contents indicating that serious damage hasoccurred It was found that osmotic stress increasedthe peroxidase activity and decreased the CAT activityin susceptible varieties of sorghum seedlings but theactivity of these enzymes increased in resistant varietyunder osmotic stress (Vardhini amp Rao 2003) Underheat stress in Lilium longiflorum plants at 37 and 42degC antioxidant enzyme activities such as POD SODAPX and CAT were stimulated However at 47 degCPOD and CAT activity was significantly decreased(Yin et al 2008) It has been reported that PODactivity in wheat leaves decreased when subjected towater stress (Baisak et al 1994) SOD and CATactivity increased in drought stressed mature leavesof Arabidopsis thaliana (Jung 2003) In this researchthe activity of enzymes studied (POD SOD CATAPX) increased when pre-treated with BR Thereason for the increase in the activity of these enzymes

may be the effects of BR on expression of biosyntheticgenes of these enzymes that resulted in increasedoxidation of harmful substrates as reported byOgweno et al (2008) Cao et al (2005) Nemhauserand Chory (2004) and Shahbaz et al (2008) Theseresults accompanied with the decrease in lipidperoxidation and high proline contents probablyrepresent a decline in ROS and an indicator ofremoval of stressful conditions by antioxidantenzymes activated by BR Similarly it has beenobserved that BR application caused an increase inCAT activity in both susceptible and resistant varietiesof sorghum seedlings under osmotic stress (Vardhiniamp Rao 2003) Furthermore the activities ofantioxidant enzymes such as SOD APX CAT andguaiacol POD were significant in EBL-treated tomatoplants under heat stress (Dhaubhadel et al 1999Ogweno et al 2008) and Mazorra et al (2002)reported that EBL and MH5 (BR analogue) stimulatedCAT activity in tomato leaf discs under heat stressApplication of 28-HBL or 24-EBL as a foliar spraywith varying levels ie 005 1 and 3 μM increasedthe catalase activity in groundnut plants withincreasing levels of BL (Vardhini amp Rao 2000) TheGR activity was investigated to address the question ofwhether 24-EBL in drought stress causes changes inthe other antioxidative enzymes It was observed atboth concentrations of 24-EBL especially at 1 μM 24-EBL concentration that GR activity was 120


424

POD1

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Control 3 day drought 5 day droughtControl

APX1

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Figures 11-13 Effects of BR and drought stress on POD CAT and APX isoforms by native PAGE Lanes 1-3 represent different levelsof BR concentration (0 001 and 1 μM)

11

12 13

indicating that 24-EBL may be effective in protectionfrom drought stress GR activity maintains the poolof glutathione in the reduced state which in turnreduces dehydroascorbate to ascorbate Increasedexpression of GR enhances tolerance to oxidativestress (Noctor amp Foyer 1998)

Exogenous applied 24-EBL enhanced SOD PODand CAT activities in salt stressed plants of wheat(Shahbaz et al 2008) Although it has been shown thatBRs can induce the expression of some antioxidantgenes and enhance the activities of antioxidantenzymes such as SOD CAT APX and POD (Ogwenoet al 2008) it is still unclear whether BRs directly orindirectly modulate the responses of plants to oxidativestress (Cao et al 2005) Nevertheless although BRsand ROS are thought to act as secondary messengersfor the induction of antioxidant defences in stressedplants (Mazorra et al 2002) the relationship betweenBRs and ROS in stress-signal transduction remainsunclear (Ogweno et al 2008)

In conclusion our results may show that theleaves of tomato plants subjected to drought stresscan endure moderate drought stress because of smallchanges in enzyme activities However enzymeactivities declined under severe drought conditionsaccompanied by a significant increase in MDAconcentration indicating serious oxidative damageby the over-accumulation of H2O2 From this result itcould be concluded that the Tomba (BB 204) varietyof tomato plant is drought sensitive because agradual loss in antioxidant protection in the leavesof plants under drought stress was observed Pre-treatment with BR can ameliorate the adverse effectsof drought stress via decreasing the oxidative damageof plant membranes possibly by the induction ofcompatible solute for osmotic adjustment and theinduction of antioxidant defence system in tomatoplants Finally the results hint that BR may in futurefind application for improving plant growth andyield in dry areas


425

Alexieva V Sergiev I Mapelli S amp Karanov E (2001) The effect ofdrought and ultraviolet radiation on growth and stress markersin pea and wheat Plant Cell Environ 24 1337-1344

Ali B Hayat S amp Ahmad A (2007) 28-Homobrassinolide amelioratethe saline stress in chickpea (Cicer arietinum L) Environ ExpBot 59 217-223

Alia B Pardha Saradhi P amp Mohanty P (1997) Involvement of prolinein protecting thylakoid membranes against free radical-inducedphotodamage J Photochem Photobiol 38 253-257

Amzallag GN amp Vaisman J (2006) Influence of brassinosteroids oninitiation of the root gravitropic response in Pisum sativumseedlings Biologia Plantarum 50 283-286

Anjum F Rishi V amp Ahmed F (2000) Compatibility of osmolytes withGibbs energy of stabilization of proteins Biochem Biophys Acta1947 75-84

Anuradha S amp Rao SSR (2001) Effect of brassinosteroids on salinitystress induced inhibition of seed germination and seedling ofrice (Oryza sativa L) Plant Growth Regul 33 151-153

Anuradha S amp Rao SSR (2003) Application of brassinosteroids to riceseeds (Oryza sativa L) reduced the impact of salt stress ongrowth prevented photosynthetic pigment loss and increasednitrate reductase activity Plant Growth Regul 40 29-32

Apel K amp Hirt H (2004) Reactive oxygen species metabolismoxidative stress and signal transduction Annu Rev Plant Biol55 373-399

Baisak R Rana D Acharya PBB amp Kar M (1994) Alterations in theactivities of active oxygen scavenging enzymes of wheat leavessubjected to water stress Plant Cell Physiol 35 489-495

Bates LS Waldren RP amp Teare ID (1973) Rapid determination of freeproline for water stress studies Plant and Soil 29 205-207

Baxter CJ Redestig H Schauer N Repsilber D Patil KR Nielsen JSelbig J Liu J Fernie AR amp Sweetlove LJ (2007) The metabolicresponse of heterotrophic arabidopsis cells to oxidative stressPlant Physiol 143 312-325

Bishop GJ amp Koncz C (2002) Brassinosteroids and plant steroidhormone signaling Plant Cell 14 97-110

Bishopp A Maumlhoumlnem AP amp Helariutta Y (2006) Signs of changeshormone receptors that regulate plant developmentDevelopment 133 1857-1869

Blokhina O Virolainen E amp Fagerstedt KV (2003) Antioxidantsoxidative damage and oxygen deprivation stress a review AnnBot 91 179-194

Borsanio O Valpuesta V amp Botella MA (2001) Evidence for a role ofsalicylic acid in the oxidative damage generated by NaCl andosmotic stress in Arabidopsis seedlings Plant Physiol 126 1024-1030

Bradford MM (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding Anal Bioch 72 248-254

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Clouse SD amp Feldmann KA (1999) Molecular genetics ofbrassinosteroid action In Sakurai A Yokota T and Clouse SD(eds) Brassinosteroids Steroidal Plant Hormones SpringerndashVerlag Tokyo Japan pp 163-189

Clouse SD amp Sasse JM (1998) Brassinosteroids essential regulators ofplant growth and development Annu Rev Plant Physiol PlantMol Biol 49 427-451

Davis PJ (2005) Plant hormones biosynthesis signal transductionaction Publisher Springer Chapter F2

Dhaubhadel S Chaudhary S Dobinson KF amp Krishna P (1999)Treatment with 24-epibrassinolide a brassinosteroid increasesthe basic thermo-tolerance of Brassica napus and tomatoseedlings Plant Mol Biol 40 333-342

Dhindsa RS Plumb-Dhindsa P amp Thorpe TA (1981) Leaf senescencecorrelated with increased levels of membrane permeability andlipid peroxidation and decreased levels of superoxide dismutaseand catalase J Exp Bot 32 93-101

Egert M amp Tevini M (2002) Influence of drought on somephysiological parameters symptomatic for oxidative stress inleaves of chives (Allium schoenoprasum) Environ Exp Bot 4843-49

Fielding JL amp Hall JL (1978) A biochemical and cytological study ofperoxidase activity in roots of Pisum sativum J Exp Bot 29 969-981

Foyer CH amp Halliwell B (1976) Presence of glutathione andglutathione reductase in chloroplasts a proposed role inascorbic acid metabolism Planta 133 21-25

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Gong JR Zhao AF Huang YM Zhang XS amp Zhang CL (2006) Waterrelations gas exchange photochemical efficiency andperoxidative stress of four plant species in the Heihe drainagebasin of northern China Photosynthetica 44 355-364

Halliwell B (2006) Reactive species and antioxidants redox biologyis a fundamental theme of aerobic life Plant Physiol 141 312-322

Haubrick LL amp Assmann SM (2006) Brassinosteroids and plantfunction some clues more puzzles Plant Cell and Environ 29446-457

Haubrick LL Torsethaugen G amp Assmann SM (2006) Effect ofbrassinolide alone and in contact with abscisic acid on controlof stomatal aperture and potassium currents of Vicia faba guardcell protoplasts Physiol Plant 128 134-143

Heath RL amp Packer L (1969) Photoperoxidation in isolatedchloroplast I Kinetics and stoichiometry of fatty acidperoxidation Arch Biochem Biophys 125 189-198

Ingram J amp Bartels D (1996) The molecular basis of dehydrationtolerance in plants Plant Physiol 47 377-403

Inze J amp Montagu MV (2000) Oxidative stress in plants TJInternational Ltd Padstow Carnawell Great Britain 321 pages

Jung S (2003) Variation in antioxidant metabolism of young andmature leaves of Arabidopsis thaliana subjected to droughtPlant Sci 166 459-466

Kamuro Y amp Takatsuto S (1999) Practical applications ofbrassinosteroids in agricultural fields In Salurai A Yokota Tand Clouse SD (eds) BrassinosteroidsndashSteroidal Plant HornonesSpringer Tokyo pp 223-241

Kavir Kishor PB Sangam S Amrutha RN Sri Laxmi P Naidu KRRao KRSS Rao S Reddy KJ amp Sreenivasulu N (2005)Regulation of proline biosynthesis degradation uptake andtransport in higher plants Its implications in plant growth andabiotic stress tolerance Cur Sci 88 424-438

Khripach VA Zhabinskii VN amp De Groot AE (2000) Twenty yearsof brassinosteroids Steroidal hormones warrant better crops forthe XXІ Century Ann Bot 86 441-447

Krishna P (2003) Brassinosteroid-mediated stress response J PlantGrowth Regul 22 289-297

Kulaeva ON Burkhanova EA Fedina AB Khokhalova VABokebayeva GA Vorbrodt HM amp Adam G (1991) Effect ofbrassinosteroids on protein synthesis and plant cellultrastructure under stress condition In Cutler HG Yokota TAdam G (eds) Brassinosteroids chemistry bioactivity andapplications ACS Symposium Series American Chemical SocietyWashington DC pp 141-155

Laemmli UK (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4 Nature 227 680-685

Li L amp Van Staden J (1998) Effects of plant growth regulators on theantioxidant system in seedlings of two maize cultivars subjectedto water stress Plant Growth Regul 25 81-87

Matysik J Alia Bhalu B amp Mohanty P (2002) Molecular mechanismsof quenching of reactive oxygen species by proline under stressin plants Curr Sci 82 525-532

Mazorra LM Nunez M Hechavarria M Coll F amp Sanchez-Blanco MJ(2002) Influence of brassinosteroids on antioxidant enzymesactivity in tomato under different temperatures Biol Plant 45593-596

McCord JM (2000) The evolution of free radicals and antioxidativestress Am J Med 108 652-654

Meidner H (1984) Class Experiments in Plant Physiology GeorgeAllen amp Unwin pp 156

Meirs S Philosophhadas S amp Aharoni N (1992) Ethylene-increasedaccumulation of fluorescent lipidndashperoxidation productsdetected during senescence of parsley by a newly developedmethod J of the American Society for Hort Science 117 128-132


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Mittler R amp Zilinskas BA (1993) Detection of ascorbate peroxidaseactivity in native gels by inhibition of the ascorbate dependentreduction of nitroblue tetrazolium Anal Biochem 212 540-546

Mittler R (2002) Oxidative stress antioxidants and stress toleranceTrends Plant Sci 7 405-410

Mittler R (2006) Abiotic stress the field environment and stresscombination Trends Plant Sci 11 15-19

Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004)Reactive oxygen gene network of plants Trends Plant Sci 9 490-498

Montoya T Nomura T Yokota T Farrar K Harrison K Jones JGDKaneta T Kamiya W Szekeres M amp Bishop GR (2005) Patternsof dwarf expression and brassinosteroid accumulation intomato reveal the importance of brassinosteroid synthesisduring fruit development Plant J 42 262-269

Nakano Y amp Asada K (1981) Hydrogen peroxide is scavenged byascorbate-specific peroxidase in spinach chloroplasts Plant CellPhysiol 22 867-880

Nakashita H Yasuda M Nitta T Asami T Fujioka S Aria Y SekimataK Takatsuto S Yamaguchi I amp Yoshida S (2003) Brassinosteroidfunctions in a broad range of disease resistance in tobacco andrice Plant J 33 887-898

Nemhauser JL amp Chory J (2004) Bring it on new insights into themechanism of brassinosteroid action J Exp Bot 55(395) 265-270

Noctor G amp Foyer CH (1998) Ascorbate and glutathione keepingactive oxygen under control Annu Rev Plant Physiol Plant Mol49 249-279

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Plewa MI Smith SR amp Wagner ED (1991) Diethyldithiocarbamatesuppress the plant activation of aromatic amines into mutagenesby inhibiting tobacco cell peroxidase Mutat Res 274 57-64

Rao SSR Vardhini BV Sujatha E amp Anuradha S (2002)Brassinosteroids ndash a new class of phytohormones Curr Sci 821239-1245

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Scandalios JG (2002) Oxidative stress responses ndash What have genomescale studies taught us Genome Biol 3 10191-10196

Schilling G Schiller C amp Otto S (1991) Influence of brassinosteroidson organ relation and enzyme activities of sugar beet plants InCutler HG Yokota and Adam G (eds) BrassinosteroidsChemistry Bioactivity and Applications ACS symp Ser 474American Chemical Society Washington DC pp 208-219

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Zhu JK (2001) Cell signaling under salt water and cold stressesCurrent Opinion in Plant Biol 4 401-406

severely damages target molecules catastrophiccascades of events set in leading to cell death (Inze ampMontagu 2000) Cells could be protected either by theendogenous molecular systems or exogenous appliedcompounds that mitigate the stress (Ingram amp Bartels1996) Capacity of plants to detoxify reactive oxygenspecies has been related to the stress tolerance ofplants (Egert amp Tevini 2002 Apel amp Hirt 2004)

Oxidative stress is a key underlying component ofmost abiotic stresses (Mittler 2002 Apel amp Hirt 2004Yin et al 2008) and a major limiting factor to plantgrowth in the field (Scandalios 2002 Blokhina et al2003 Mittler 2006) Production of reactive oxygenspecies (ROS) and other radicals increasesdramatically during abiotic stress conditions andoxidative stress occurs when the rate of ROSproduction outstrips the capacity of antioxidantsystems to detoxify them (McCord 2000 Mittler2002 Mittler et al 2004) The result is oxidativedamage to key biomolecules such as proteins lipidsand DNA leading to cellular dysfunction andultimately cell death (Wagner et al 2004 Halliwell2006 Baxter et al 2007) To avoid oxidative damageplants invoke a molecular response that allows themto cope with and adapt to the oxidative stresssituation Many stress-related genes have beenidentified and the list of antioxidant enzymeshormones and metabolites present in plantscontinues to be extended (Nakashita et al 2003 Caoet al 2005) One type of these compounds that haveantioxidative characteristics is brassinosteroids (BRs)(Bishop amp Koncz 2002 Haubrick amp Assmann 2006)

BRs are common plant-produced compounds thatcan function as growth regulators (Khripach et al2000 Davis 2005 Bishopp et al 2006) In addition ithas been suggested that BRs could be included in thecategory of phytohormones (Clouse amp Feldmann1999 Haubrick amp Assmann 2006) Exogenousapplication of BRs may influence a range of diverseprocesses of growth and development in plants (Caoet al 2005 Clouse amp Sasse 1998 Yu et al 2004Montoya et al 2005) In addition it is becoming clearthat BRs interact both negatively and positively withother major signalling pathways including thoseregulated by auxin and ethylene (Amzallage ampVaiseman 2006 Haubrick et al 2006) It is clear thatBRs provide protection against a number of abiotic

stresses (Sasse 1999 Rao amp Vardhini 2002 Krishna2003 Nemhauser 2004) Treatment with BRsenhanced the growth of wheat (Sairam 1994) Frenchbean (Upreti amp Murti 2004) and sugar beet plants(Schilling 1991) under drought stress Application ofBRs improved tolerance against salt in rice (Anuradhaamp Rao 2001 2003 Oumlzdemir et al 2004) groundnut(Vardhini amp Rao 1997) wheat (Sairam et al 2005)and chickpea (Ali et al 2007) It was reported that BLinduced thermotolerance in tomato (Dhaubhadel etal 1999 Mazorra et al 2002 Ogweno et al 2008)rice (Wang amp Zang 1993) and brome grass (Wilen etal 1995) Moreover BRs increased tolerance againsthigh temperatures in Brassica napus (Singh amp Shono2005) and brome grass (Wilen et al 1995) Onemechanism that may be involved in the resistance tomany types of stress is the increased activity of theantioxidant pathway Several studies have shown thatBRs alter the antioxidant capacity of plants understress conditions (Kamuro amp Takatsuto 1999Yardanova 2004 Yin et al 2008) The present studywas an attempt to carry out investigations of the effectof brassinolide on the water stress responses of tomatoplants with the aim to elucidate possible mechanismsthat might be involved in the BR-promotedantioxidant responses to drought

Abbreviations BRndashBrassinosteroid MDAndashMalondialdehyde TBAndashThiobarbituric Acid TCAndashTrichloroacetic Acid EBL-EpibrassinolideHBLndashHomobrassinolide CATndashCatalase PODndashPeroxidase APXndashAscorbate peroxidase ROSndashReactive Oxygen Species SOD-Superoxide dismutaseH2O2- peroxide hydrogen NBT- ρ-nitro bluetetrazolium chloride EDTA-Ethylendiaminetetraacetic acid GR- Glutathione reductase

Materials and methods24-Epibrassinolide (EBL C28H48O6 MW = 480)

was purchased from Sigma (USA) Tomato(Lycopersicon esculentum Mill var Tomba (BB204))seeds were used in this study Seeds were sown inplastic pots containing sand loam and peat (211)When 4 fully expanded leaves appeared (the tomatoplants were 1 month old) the tomato was left to growin 2618 degC and 168 h (lightdark) conditions in agrowth chamber Hoaglandlsquos nutrient solutions were


418














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(mg

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11

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11

12 13





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1 μM BR0 μM BR

0

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11

12 13





425

















References


426
































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424

POD1


POD3POD2


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11

12 13





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References


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the effects of brassinosteroid on the induction of...

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