This article was downloaded by: [Dr Ali Ghasemzadeh]On: 02 February 2014, At: 01:00Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK

Acta Agriculturae Scandinavica, Section B - Soil &Plant SciencePublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/sagb20

Mitigation of salt-induced oxidative damage inChinese kale (Brassica alboglabra L.) using ascorbicacidAmin Tayebi-Meigoonia, Yahya Awanga, Alan R. Biggsb, Rosli Mohamada, Babak Madania &Ali Ghasemzadeha

a Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia,Selangor, Malaysiab Division of Plant and Soil Sciences, Division of Plant and Soil Sciences, Davis College ofAgriculture, Natural Resources and Design, Western Virginia University, Morgantown, WV,USAPublished online: 20 Jan 2014.

To cite this article: Amin Tayebi-Meigooni, Yahya Awang, Alan R. Biggs, Rosli Mohamad, Babak Madani & AliGhasemzadeh , Acta Agriculturae Scandinavica, Section B - Soil & Plant Science (2014): Mitigation of salt-inducedoxidative damage in Chinese kale (Brassica alboglabra L.) using ascorbic acid, Acta Agriculturae Scandinavica, Section B -Soil & Plant Science, DOI: 10.1080/09064710.2013.869347

To link to this article: http://dx.doi.org/10.1080/09064710.2013.869347

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose ofthe Content. Any opinions and views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be reliedupon and should be independently verified with primary sources of information. Taylor and Francis shallnot be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and otherliabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

ORIGINAL ARTICLE

Mitigation of salt-induced oxidative damage in Chinese kale(Brassica alboglabra L.) using ascorbic acid

Amin Tayebi-Meigoonia*, Yahya Awanga*, Alan R. Biggsb, Rosli Mohamada, Babak Madania andAli Ghasemzadeha

aDepartment of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, Selangor, Malaysia; bDivision ofPlant and Soil Sciences, Division of Plant and Soil Sciences, Davis College of Agriculture, Natural Resources andDesign, Western Virginia University, Morgantown, WV, USA

(Received 9 July 2013; accepted 18 November 2013)

Salinity could become a threat to hydroponically grown plants due to the differential ion uptake process by thecrop itself and/or using low quality irrigation water containing non-essential ions such as sodium and chloride.This study was conducted to determine the interactive effects of salinity and foliar application of ascorbic acid(AsA) on the survival capacity of Chinese kale plants. Three-week-old plants at the 4-leaf stage were exposed tosalinity by application of 0, 50 and 100 mM NaCl for 14 days. The activities of antioxidant enzymes, catalase(CAT), peroxidase (POX) and ascorbate peroxidase (APX) along with changes in the accumulation of proline,lipid peroxidation and chlorophyll content in leaves were estimated. Salinity induced obvious changes in theactivities of antioxidant enzymes, lipid peroxidation, proline accumulation and chlorophyll content in the leaftissues. Exogenous application of AsA (5 and 10 mM) to stressed plants resulted in a significant increase (P <0.05) in APX activity and reduction in the activities of antioxidant enzymes (CAT and POX) as well as adecrease in the contents of stress-induced proline and malondialdehyde (MDA). Higher chlorophyll contentwas also recorded in stressed plants using AsA. The inherent capability of AsA to scavenge free radicals mayhave assisted the plant using the lower oxidative damages. The results of this study showed that AsA treatmentwould reduce the deleterious effects of salt-induced oxidative stress and improve plant tolerance to salt stress.

Keywords: antioxidant enzymes; chlorophyll; lipid peroxidation; NaCl salt stress; proline; protein

Introduction

Salinity is an important environmental constraint tocrop production worldwide. All major processes in-cluding photosynthesis, protein synthesis, lipidmetabolism and energy conservation mechanismsare affected by salt stress (Parida & Das 2005). Saltstress, as well as other types of environmental con-straints, induces formation of reactive oxygen species(ROS), including superoxide anion, hydrogen per-oxide and hydroxyl radicals (Zhu 2000).Various sites within the chloroplast can activate

oxygen (McKersie & Leshem 1994). Typically, ROScan be generated by direct transfer of excitationenergy from chlorophyll which converts the tripletoxygen to singlet form (McKersie & Leshem 1994;

Asada 2006) or through Mehler reaction by mono-valent oxygen reduction of photosystem I (PSI) underconditions where nicotinamide adenine dinucleotidephosphate (NADP) is limiting (McKersie & Leshem1994; Asada 1999). These aggressive free radicalscan disturb metabolic functions with damage tomembrane lipids, proteins and nucleic acids (Noctor& Foyer 1998).Plants employ various defence systems to prevent or

overcome oxidative damage, which could involve bothenzymatic and non-enzymatic constituents (Sekmenet al. 2007; Ashraf & Ali 2008; Manchanda & Garg2008). Increase in the capacity of the antioxidantdefence system in response to an increase in oxygenradicals’ generation under salinity has been reported

*Corresponding authors. Email: amin_ir@hotmail.com; yahya_awg@putra.upm.edu.my

Acta Agriculturae Scandinavica, Section B - Soil & Plant Science, 2014http://dx.doi.org/10.1080/09064710.2013.869347

© 2014 Taylor & Francis

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014

(Sekmen et al. 2007; Ashraf & Ali 2008). However, theformation of ROS can enhance to an extent that endo-genous protective activity may become inadequate.Several studies have described different associa-

tions of salt stress and endogenous levels of bothhydrophilic and lipophilic antioxidants such asascorbate and tocopherols (Shalata & Neumann2001). Also, induction of antioxidant enzymes activ-ity, e.g. superoxide dismutase (SOD), ascorbateperoxidase (APX), catalase (CAT) and peroxidase(POX), in association with salt tolerance comparedto salt sensitivity has been described (Sekmenet al. 2007).SOD as a primary scavenger converts superoxide

(O2•) to oxygen (O2) and hydrogen peroxide

(H2O2). Decomposition of hydrogen peroxide islater driven by ascorbate peroxidase, in associationwith monodehydroascorbate reductase, dehydroas-corbate reductase and glutathione reductase (Asada1999). APX functions by utilizing ascorbate as itsspecific electron donor to convert H2O2 to water,with the concomitant generation of monodehydroas-corbate. Monodehydroascorbate is regenerated toascorbic acid (AsA) by monodehydroascorbatereductase, or is disproportionated non-enzymaticallyto AsA and dehydroascorbate (Asada 2006). CATand POX also have a key role to scavenge hydrogenperoxide and decompose H2O2 to H2O and O2

(Asada 2006; Sekmen et al. 2007).AsA is a small water-soluble molecule found in

abundance on plants (Huang et al. 2005). Ascorbatereacts with superoxide anions generated through theMehler reaction and photorespiration. Therefore,ascorbate may serve a physiologically similar role toSOD (McKersie & Leshem 1994). Ascorbate is animportant compound contributing to plant growthand developmental processes such as cell divisionand cell wall expansion (Pignocchi & Foyer 2003).Ascorbate acts as a key substrate in coordinationwith glutathione, α-tocopherol and numerous anti-oxidant enzymes to counteract H2O2 which iscatalyzed by APX (Huang et al. 2005). Furthermore,ascorbate functions to regenerate membrane-boundantioxidants, such as α-tocopherol, and detoxifiesperoxyl radicals and singlet oxygen (McKersie &Leshem 1994; Noctor & Foyer 1998; Asada 1999).Ascorbate has also been found to act as a cofactor

of violaxanthin de-epoxidase, an enzyme that imple-ments the conversion of violaxanthin to zeaxanthinin the xanthophyll cycle under excess light, which isimplicated in non-photochemical quenching ofexcess excited energy in photosystem II (PSII)(Demmig-Adams & Adams 1990; Eskling et al.1997). Hence, ascorbate involvement is crucial toscavenge oxygen-free radicals generated in

photosynthesis and to dissipate excess energy of thelight-harvesting complex (Demmig-Adams & Adams1992; Niyogi 1999). Oxidative stress, indicated bythe level of hydrogen peroxide, increased moreseverely in ascorbate-deficient vtc−1 mutant com-pared to the wild type of Arabidopsis thaliana undersalt stress (Huang et al. 2005). Conversely, repletionof the AsA pool in plant exposed to salinity usingexogenous application of ascorbate reduced deleteri-ous effects of oxidative stress and improved resist-ance to salt stress in tomato, rapeseed and wheat(Shalata & Neumann 2001; Dolatabadian et al.2008; Athar et al. 2009).Based on the findings on tomato seedlings,

enhanced survival capacity of seedlings exposed to9 h of severe salt stress was likely provided throughAsA association with antioxidant activity and lowerlipid peroxidation (Shalata & Neumann 2001).Higher adaptation to salt stress in rapeseed plantsalso has been suggested to be linked with theprotective role of AsA on photosynthetic pigmentsand alleviation of salt-induced oxidative damages(Dolatabadian et al. 2008). It has also been theorizedthat lower adverse effects of salinity on wheat growthwas coupled with exogenous AsA potential fortriggering antioxidant activity, enhancing AsA endo-genesis and photosynthetic capacity as well as main-taining ion homeostasis (Athar et al. 2008, 2009).Plants grown in closed-loop hydroponics may

encounter difficulties such as salt accumulation andnutritional imbalances in the recirculating nutrientsolution that are caused by either the differential ionuptake by the crops or mostly the use of irrigationwater containing non-essential ions such as sodiumand chloride. The Brassica vegetables such as Chi-nese kale which could be successfully planted inhydroponics system are important in terms of nutri-ent quality in human diet (Kristal & Lampe 2002).Hence, understanding the physiological effects ofsalinity on plants and attempts to increase salttolerance are crucial. No attention has been devotedon the effects of salinity and exogenous AsA on thedefence mechanism and particularly antioxidantdefence system of Chinese kale. Therefore, changesin the activity of antioxidant enzymes includingCAT, POX and APX involved in the H2O2 neutral-ization process were assayed in crude leaf extracts ofChinese kale plants. Changes in malondialdehyde(MDA) content which represents the size of stressand/or capability of the antioxidant defence systemwas also examined. The study was designed toexamine the effect of foliar application of ascorbatefor preventing cell damage by altering the ROSneutralization system and improving salt tolerance.

2 A. Tayebi-Meigooni et al.

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014

Materials and methods

Plant materials and growth conditions

The seeds of Chinese kale (Brassica oleracea var.alboglabra) cv. “Standard Kailan” (11–33121) wereobtained from a local supplier in Klang Valley,Malaysia. The seeds were sown in trays maintainedwith water spray until seedling emergence. Youngseedlings in the seed trays were placed in a plasticfilm pool and irrigated with nutrient solution con-taining essential elements (Table 1). The experimentwas conducted in a greenhouse at 32/22°C day/nighttemperature, 12 h of photoperiod and 70–80%relative humidity.

Salt stress and AsA treatments

Fifteen-day-old seedlings of uniform size were trans-planted in closed-loop hydroponic system and fedwith a similar nutrient solution. Salt stress wasinitiated 21 days after germination at the 4-leaf stage.Plants were exposed to salt stress, supplied throughthe basic nutrient solution supplemented with threelevels of salt (S: 0, 50 and 100 mM NaCl) for 14days. The salt was added gradually to avoid osmoticshock. A day after salt treatment, foliar application ofAsA solutions (A: 0, 5 and 10 mM) was initiated.Tween−20 (two drops per liter) was added to theAsA solutions to improve adhesiveness of the appliedsolution to the leaves.Treatments comprised of following controls: (i) no

NaCl + no AsA (S0A0), (ii) no NaCl + 5 mM AsA(S0A1), (iii) no NaCl + 10 mM AsA (S0A2), (iv) 50mM NaCl + no AsA (S1A0), (v) 50 mM NaCl +5 mM AsA (S1A1), (vi) 50 mM NaCl + 10 mM AsA(S1A2), (vii) 100 mM NaCl + no AsA (S2A0), (viii)100 mM NaCl + 5 mM AsA (S2A1) and (ix) 100mM NaCl + 10 mM AsA (S2A2).

Sampling

Five plants were sampled from each pot at two weeksafter salt stresses were imposed. Leaf samples (newfully expanded) were frozen on site with liquidnitrogen and retained at –80°C for further biochem-ical analysis.

Extraction for protein and enzyme assays

The frozen samples (0.2 g) were homogenized with3-ml ice-cold 50 mM potassium phosphate buffer(pH 7.8) and centrifuged at 18000 × g for 20 min at4°C. To extract APX, 2 mM ascorbate was includedinto the extraction buffer. The supernatants were usedas crude extract in the protein and enzyme assays.

Protein assay

Total protein content was determined using bovineserum albumin (BSA) as a standard following themethod described by Bradford (1976).

Assay of antioxidant enzymes

CAT activity was estimated according to the methodof Cakmak et al. (1993). The reaction mixture (1 ml)was made up of enzyme extract (100 µl) along with25 mM potassium phosphate buffer (pH 7.0) and10 mM H2O2. The decline in absorbance at 240 nmas a consequence of H2O2 decomposition was mon-itored for 1 min using a UV-visible spectrophotometer(Model Varian Cary 50 UV-Vis Spectrophotometer,USA). CAT activity was calculated using the extinc-tion coefficient for H2O2 (39.4 mM–1 cm–1).APX activity was determined following the

method of Webb and Allen (1995) with a slightmodification. The assay mixture contained 50 mMphosphate buffer (pH 7.0), 0.2 mM EDTA, 0.25mM H2O2, 0.5 mM sodium ascorbate and enzymeextract (100 µl) in a total volume of 1.0 ml. Changesin optical density of the reacting samples weremonitored for 1 min by a decrease in absorbance at290 nm. The APX activity was expressed as unitsmg–1 protein (extinction coefficient 2.8 mM–1 cm–1).POX activity was assayed by guaiacol oxidation

method as described by Ghanati et al. (2002). Thefinal volume (3.0 ml) of the reaction mixture con-tained 100 µl crude extract as well as 50 mMpotassium phosphate buffer (pH 7.0), 28 mMguaiacol and 10 mM H2O2. The increase in absor-bance at 470 nm was recorded every 30 s. The APXactivity was expressed as units per mg solubleprotein (extinction coefficient 26.6 mM–1 cm–1).

Table 1. Nutrients composition of the solution used in the study.

Nutrient (mg L–1)

N P K Ca Mg S Fe Mn B Cu Zn Mo

250 67 239 160 30 80 3 0.62 0.44 0.02 0.11 0.048

Acta Agriculturae Scandinavica, Section B - Soil & Plant Science 3

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014

Determination of lipid peroxidation

Lipid peroxidation was estimated by the level ofMDA production based on the thiobarbituric acid(TBA) method as described by Du and Bramlage(1992). Fresh leaf samples (0.25 g) were homogenizedin 5 ml of 0.1% (w/v) trichloroacetic acid (TCA)solution and centrifuged at 15000× g for 15 min.The supernatant (1.0 ml) was added to 4 ml 0.5%(w/v) TBA in 20% TCA (w/v). The mixture wasthen heated in boiling water for half an hour andimmediately cooled to stop the reaction. The absor-bance of the resulting supernatant was recorded at532 nm and corrected for non-specific turbidity bysubtracting the absorbance at 600 nm. MDA con-centrations were calculated from the extinction coef-ficient of 155 mM–1 cm–1 using the following formula.

MDAðnmol/g fresh weightÞ¼ ½ðA532 � A600Þ/155� � 103 � dilution factor ð1Þ

Determination of proline

Proline content was measured based on Bates et al.(1973). Samples of leaves (0.2 g) were homogenizedin 3 ml sulphosalicylic acid (3% w/v), and theextracts filtered. Equal amounts of glacial aceticacid and ninhydrin solution were added to 2 ml ofthe extract and then were incubated for 1 h at 95°C.The cooled mixture was treated with 4 ml of tolueneand incubated for 10 min. Absorbance of thechromophore-containing phase at 520 nm wasrecorded. Proline content was estimated using astandard curve and was expressed as µmol g–1 freshweight (FW).

Determination of chlorophyll pigments

Chlorophyll pigments (Chlorophyll a and b) wereextracted in 95% (v/v) acetone and determinedfollowing the method described by Lichtenthalerand Buschmann (2001). The contents of Chloro-phyll a and b were calculated using the followingrelationships:

Chlorophyll a ðmg/mlÞ¼ 11.24 A661.6 � 2.04 A644.8 ð2Þ

Chlorophyll b ðmg/mlÞ¼ 20.13 A644.8 � 4.19 A661.6 ð3Þ

Determination of relative water content

The relative water content (RWC) was determinedon fresh leaf samples using the formula described by

Smart and Bingham (1974).

RWC ¼ fresh weight � dry weightturgid weight � dry weight

� 100 ð4Þ

Experimental design and data analysis

A randomized complete block design with fourreplications was used. Data were analyzed using theSAS 9.1 software (SAS Institute Inc. 2004). Meanscomparison was conducted using the Duncan’s mul-tiple range test (DMRT) at 5% level of probability.

Results

All measured parameters were affected by salinityand the foliar application of AsA had a significanteffect on those traits, except for leaf RWC, chloro-phyll b concentration and CAT activity (Table 2).The results showed there was no significant differ-ence between AsA levels (5 and 10 mM) in term oftheir effects on variables. The effects of salinity onlipid peroxidation (MDA) as well as the activities ofCAT and POX were dependent on AsA levels asindicated by the significant NaCl × AsA interactions.

Antioxidant enzymes and protein content

The interaction of NaCl salinity with AsA levels wassignificant for both CAT and POX activities (P ≤0.05). CAT activity was greatest for the highestsalinity in the absence of AsA while application ofAsA over 100 mM NaCl salinity reduced CATactivity about 22 and 19% for 5 and 10 mM AsA,respectively. CAT activity was almost unchanged atboth 0 and 50 mM NaCl salinity with either AsApresence or AsA absence (Figure 1A).POX activity was at the minimum level for the

non-saline condition whether AsA was applied ornot. Since POX activity was boosted with increasingsalinity, there was an obvious tendency for reductionin POX activity with higher AsA concentration. POXactivity was at the maximum point as salinity reachedat 100 mM NaCl in the lack of AsA, althoughincreasing AsA from 0 to 5 and 10 mM resulted ina significant suppression in the activity of POX by19 and 26% in leaves, respectively (Figure 1B).Salt-stressed plants showed higher activity of APX

enzyme in leaf tissues compared to non-stressedplants (Figure 2A). APX activity was progressivelyraised from 2.02 to 2.86 fold with 50–100 mM NaCltreatments. APX activity remained approximatelyconstant for non-stressed plants with the applicationof AsA, while APX activity increased followingAsA application by up to 35 and 21% in plants

4 A. Tayebi-Meigooni et al.

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014

treated with 50 and 100 mM NaCl, respectively(Figure 2B).Leaf protein content was significantly decreased in

plants subjected to salinity, relative to control plants.It was decreased by 11.5–31.5% for the 50–100 mMNaCl salinity treatments, respectively (Figure 2C).Soluble protein content in salinity-exposed plantswas increased with the application of AsA byapproximately 5–10% compared to those not treatedwith AsA (Figure 2D). Application of AsA did notaffect the leaf protein content in non-stressed plants.

Lipid peroxidation

The level of lipid peroxidation in leaf tissues isreflected by MDA content. There was a significantinteraction between salinity and AsA on the level ofMDA (Figure 3). The smallest amount of MDA wasobserved at the lowest salinity regardless of AsAapplication. The MDA level was boosted by increas-ing salinity from 0 to 50 and 100 mM NaCl in theabsence of AsA, while its production was signifi-cantly (P < 0.05) inhibited (about 22%) as AsA levelreached 5 and 10 mM.

Proline accumulation

Accumulation of free proline in the leaves of plantsexposed to salinity was significantly amplified. Incomparison to the control treatment, increases inproline were about 8.3–16.3 fold for 50 and 100 mMsalinity treatments, respectively (Figure 4A). Theascorbate application resulted in a 15 and 12.5%reduction of proline content in plants subjected to50 and 100 mM salinity, respectively. Exogenousapplication of AsA did not affect the proline contentof non-stressed plants (Figure 4B).

Relative water content

Leaf RWC was adversely affected by increase insalinity. RWC was reduced by 11.8 and 17.1% asNaCl concentration increased from 0 to 50 and 100mM concentrations, respectively. Application of AsAdid not change RWC in stressed plants. RWC ofnon-stressed plants also did not show any significantchanges when treated with AsA (Figure 4C).

Photosynthetic pigments

Leaf chlorophyll content (a and b) was significantlyaffected in plants exposed to salinity (Figure 5A and5B). Total chlorophyll was reduced by approxi-mately 14 and 30% with the 50 and 100 mM NaCltreatments, respectively (Figure 5C). Foliar applica-tion of ascorbate (100 mM) resulted in a 16.6%T

able2.

Meansqua

resextractedfrom

analyses

ofvarian

ce(A

NOVAs)

oftheda

taforRW

C,c

ontentsof

free

proline,

MDA,p

rotein,c

hlorop

hylls

andactivities

ofCAT,P

OX

andAPX

ofChine

sekale

unde

rdifferen

tNaC

lsalin

ityan

dAsA

treatm

ents

aswellas

theirinteractions.

Chlorop

hyll

Sou

rceof

variation

dfProlin

ea

ba+b

RW

CM

DA

Protein

POX

CAT

APX

Rep

lication

362

.33n

s0.00

02ns

0.00

02ns

0.00

06ns

10.61n

s0.43

36ns

0.38

95ns

0.06

79ns

0.00

19ns

0.28

54ns

NaC

l2

4472

0.7*

**0.14

4***

0.02

3***

0.28

1***

915.3*

**35

.51*

**11

.099

***

20.26*

**0.63

4***

46.37*

**AsA

223

7.77

*0.01

9*0.00

12ns

0.02

90*

18.422

ns6.00

34**

*0.76

0*0.33

0*0.00

07ns

1.77

1*NaC

l×AsA

499

.497

ns0.00

78ns

0.00

065n

s0.01

26ns

11.522

ns1.53

8*0.36

8ns

0.28

8*0.07

14*

0.63

8ns

Error

3591

859.1

0.47

930.06

920.87

5721

87.8

102.28

31.097

44.587

2.03

4110

7.89

CV

(%)

10.78

7.33

7.25

6.89

3.99

9.87

6.34

8.36

9.17

7.25

Notes:ns,no

tsign

ificant.

*P<0.05

,**

*P≤

0.00

1.

Acta Agriculturae Scandinavica, Section B - Soil & Plant Science 5

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014

Figure 1. Effects of NaCl salinity × ascorbic acid interaction on catalase (CAT) activity (A) and peroxidase (POX) activity(B) in the leaves of B. oleracea var. alboglabra (means ± SE, n = 4).Note: Bars with different letters indicate significant differences according to Duncan’s multiple range test (DMRT) atP < 0.05.

Figure 2. Effects of NaCl salinity and ascorbic acid (AsA) on ascorbate peroxidase (APX) activity (A and B) as well ascontent of protein (C and D) in the leaves of B. oleracea var. alboglabra (means ± SE, n = 4).Notes: Bars with different letters indicate significant differences according to Duncan’s multiple range test (DMRT) atP < 0.05. FW, fresh weight.

6 A. Tayebi-Meigooni et al.

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014

higher chlorophyll a content and a 18.7% highertotal chlorophyll (a + b) content in stressed plantscompared to plants that were not treated withascorbate (Figure 5D and 5E). Chlorophyll b didnot differ markedly with the application of ascorbatein stressed plants.

Discussion

High salinity as well as other abiotic stresses causesmolecular damage to cellular and sub-cellular con-stituents of plants either directly or indirectlythrough the generation of ROS. In the present study,CAT, APX and POX activities in leaves of plantsexposed to salinity were significantly increased. Theassayed enzymes are considered as oxygen-free rad-ical scavengers. Following the conversion of super-oxide (O2

�) by SOD to another equally toxic one(H2O2), CAT and POX eliminate H2O2 in company

Figure 4. Effects of NaCl salinity and ascorbic acid (AsA) on proline content (A and B), as well as effects of NaCl salinityon relative water content (RWC) (C) in the leaves of B. oleracea var. alboglabra (means ± SE, n = 4).Notes: Bars with different letters indicate significant differences according to Duncan’s multiple range test (DMRT) atP < 0.05. FW, fresh weight.

Figure 3. Effects of NaCl salinity × ascorbic acid (AsA)interaction on malondialdehyde (MDA) content in theleaves of B. oleracea var. alboglabra (means ± SE, n = 4).Note: Bars with different letters indicate significant differ-ences according to Duncan’s multiple range test (DMRT)at P < 0.05.

Acta Agriculturae Scandinavica, Section B - Soil & Plant Science 7

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014

with APX which has greater affinity to remove H2O2

by using ascorbate as the specific electron donor(Hegedus et al. 2001).Based on the results, formation of oxygen radicals

directly or indirectly induced by salt stress leads to

oxidative damage such as lipid peroxidation. LimitedCO2 availability in leaves due to stomatal closureunder salt-stressed conditions leads to a decline incarbon fixation, which exposes the chloroplasts toexcessive excitation energy leading to increased

Figure 5. Effects of NaCl salinity on content of chlorophyll a (A), chlorophyll b (B) and total chlorophyll (C), as well aseffects of ascorbic acid (AsA) on chlorophyll a (D) and total chlorophyll (E) contents in the leaves of B. oleracea var.alboglabra (means ± SE, n = 4).Notes: Bars with different letters indicate significant differences according to Duncan’s multiple range test (DMRT) atP < 0.05. FW, fresh weight.

8 A. Tayebi-Meigooni et al.

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014

formation of free radicals and higher oxidative stress(Gossett et al. 1994a, 1994b).Similar increases in the activities of CAT and APX

were observed in salt-tolerant tomato, sugar beet andrice (Shalata & Tal 1998; Bor et al. 2003; Demiral &Türkan 2005). Similar increases in the activity ofPOX have also been reported in Plantago sp. (Sek-men et al. 2007). There was an obvious decrease inthe activities of CAT and POX when AsA wasapplied to the leaves of the stressed plants. Thisreduction in the activity of enzymes with the applica-tion of AsA can be attributed to the capability ofascorbate to eliminate free radicals. Higher APXactivity with exogenous AsA was also observed in thestressed plants, which can be explained by APXaffinity for utilizing ascorbate as its specific electrondonor.Ascorbate acts directly to detoxify oxygen-free

radicals with or without enzyme catalysts and indir-ectly scavenge them by regenerating tocopherol tothe reduced form (McKersie & Leshem 1994).Ascorbate is oxidized by oxygen-free radicals toform monodehyderoascorbate and dehydroascorbate(Miyake & Asada 1992; Noctor & Foyer 1998).Stress conditions may lead to ascorbate depletionfollowed by oxidative damage intensification.Plants may suffer oxidative stress by ROS reaction

with target substances, such as proteins, lipids and/ornucleic acids. MDA, as final product of lipidperoxidation, is widely used as an indicator toevaluate the extent of oxidative stress (Lin & Kao2000; Tayebimeigooni et al. 2012), and reflects cellmembrane stability under stress. Higher membranestability is considered to be correlated with abioticstress tolerance (Premachandra et al. 1992). In thecurrent study, accumulation of MDA was increasedunder salt stress. A reduction in the protein contentof plants subjected to salinity was also observed.Reduction of protein content was probably due tothe induction of protein degradation by oxygen-freeradicals (Davies 1987). Application of AsA partiallyinhibits these processes via AsA affinity to scavengeROS (Noctor & Foyer 1998). Nevertheless, theactual inhibition mechanism of AsA on lipid perox-idation is still unclear (Dolatabadian et al. 2008).Accumulation of proline, a highly water-soluble

amino acid, is an accepted metabolic phenomenon ofplants to salt-stress adversity (Hasegawa et al. 2000;Nandwal et al. 2000). Proline as an osmoprotectant isbelieved to play a crucial role in osmoregulation andosmotolerance processes (Daneshmand et al. 2010).It is also known to act as a protein-compatible hyd-rotrope, plasma membrane protector and hydroxylradical scavenger (Dolatabadian et al. 2008). In thepresent study, accumulation of proline was progres-sively induced in the leaves of stressed plants, and

exogenous application of AsA prevented biosynthesisof extra proline probably due to the scavenging ofROS by exogenous AsA. These findings arein agreement with the results of Dolatabadianet al. (2008).Oxidative stress causes cellular senescence due to

the loss of chlorophyll and proteins along withdecreased membrane permeability, which restrictsphotosynthetic capacity (Thompson et al. 1987).Increase in free radical formation in chloroplastsleads to the destruction of chlorophyll molecules,and consequently a reduction in photosynthesis andgrowth.Aggressive singlet oxygen atoms and superoxide

anions mainly cause the destruction of double bond-containing compounds such as unsaturated fattyacids and chlorophylls, followed by damage to thechloroplast membrane system and photosyntheticreaction centers (Zhang et al. 2003), which in turnmay result in the release of chlorophylls from thethylakoid membranes. Under such circumstances,rapid chlorophyll degradation to avoid harmfuleffects of its photodynamic action to cellular compo-nents is needed (Takamiya et al. 2000), and failurein chlorophyll degradation results in higher quantityof ROS production to an extent where the detoxi-fication capability of antioxidant systems maybecome inadequate or even overridden. Biosynthesisof chlorophyll has been inhibited in plants due tometal and salt stress (Sinha et al. 2003). The key roleof AsA in detoxification and neutralization of freeradicals by prevention of superoxide anion andsinglet oxygen activity is to enhance or retainchlorophyll content.Results of the present study have shown that the

survival capacity of Chinese kale was enhanced bythe exogenous application of ascorbate under salineconditions. Increase in salt stress tolerance is attrib-uted to the anti-oxidative capability of AsA which isreflected by a partial alleviation of membrane damage(lipid peroxidation) together with higher chlorophyllretention and inferior and/or greater antioxidantenzymes activity, suggesting that usage of ascorbatecan mitigate detrimental effects of oxygen-free radi-cals and promote plant resistance as part of the plantprotection system.

AcknowledgementsThe authors are grateful to the Research ManagementCentre of Universiti Putra Malaysia for financing andsupporting this work.

References

Asada K. 1999. The water–water cycle in chloroplasts:scavenging of active oxygen and dissipation of excess

Acta Agriculturae Scandinavica, Section B - Soil & Plant Science 9

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014

photons. Annu Rev Plant Physiol Plant Mol Biol.50:601–639.

Asada K. 2006. Production and scavenging of reactiveoxygen species in chloroplasts and their functions.Plant Physiol. 141:391–396.

Ashraf M, Ali Q. 2008. Relative membrane permeabilityand activities of some antioxidant enzymes as the keydeterminants of salt tolerance in canola (Brassicanapus L.). Environ Exp Bot. 63:266–273.

Athar HR, Khan A, Ashraf M. 2008. Exogenously appliedascorbic acid alleviates salt-induced oxidative stress inwheat. Environ Exp Bot. 63:224–231.

Athar HR, Khan A, Ashraf M. 2009. Inducing salt tolerancein wheat by exogenously applied ascorbic acid throughdifferent modes. J Plant Nutr. 32:1799–1817.

Bates LR, Waldren RP, Teare ID. 1973. A rapid deter-mination of free proline for water stress studies. PlantSoil. 39:205–207.

Bor M, Ozdemir F, Türkan I. 2003. The effect of saltstress on lipid peroxidation and antioxidants in leavesof sugar beet Beta vulgaris L. and wild beet Betamaritima L. Plant Sci. 164:77–84.

Bradford M. 1976. A rapid and sensitive method forthe quantitation of protein utilizing the principle ofprotein-dye binding. Annu Rev Biochem. 72:248–254.

Cakmak I, Strbac D, Marschner H. 1993. Activities ofhydrogen peroxide scavenging enzymes in germinatedwheat seeds. J Exp Bot. 44:127–132.

Daneshmand F, Arvin MJ, Kalantari KM. 2010. Physio-logical responses to NaCl stress in three wild speciesof potato in vitro. Acta Physiol Plant. 32:91–101.

Davies KJA. 1987. Protein damage and degradation byoxygen radicals. I. General aspects. J Biol Chem.262:9895–9901.

Demiral T, Türkan I. 2005. Comparative lipid peroxida-tion, antioxidant systems and proline content in rootsof two rice cultivars differing in salt tolerance. EnvironExp Bot. 53:247–257.

Demmig-Adams B, Adams WW. 1990. The carotenoidszeaxanthin and high-energy-state quenching of chloro-phyll fluorescence. Photosynth Res. 25:187–198.

Demmig-Adams B, Adams WW. 1992. Photoprotectionand other responses of plants to high light stress.Annu Rev Plant Physiol Plant Mol Biol. 43:599–626.

Dolatabadian A, Sanavy SAMM, Chashmi NA. 2008. Theeffects of foliar application of ascorbic acid (VitaminC) on antioxidant enzymes activities, lipid peroxida-tion and proline accumulation of canola (Brassicanapus L.) under conditions of salt stress. J AgronCrop Sci. 194:206–213.

Du Z, Bramlage WJ. 1992. Modified thiobarbituric acidassay for measuring lipid oxidation in sugar-rich planttissue extracts. J Agr Food Chem. 40:1566–1570.

Eskling M, Arvidsson PO, Akerlund HE. 1997. Thexanthophylls cycle, its regulation and components.Physiol Plantarum. 100:806–816.

Ghanati F, Morita A, Yokota H. 2002. Induction ofsuberin and increase of lignin content by excess boronin tobacco cell. Soil Sci Plant Nutr. 48:357–364.

Gossett DR, Millhollon EP, Lucas MC. 1994a. Antiox-idant response to NaCl stress in salt tolerant and saltsensitive cultivars of cotton. Crop Sci. 34:706–714.

Gossett DR, Millhollon EP, Lucas MC, Banks SW,Marney MM. 1994b. The effects of NaCl on anti-oxidant enzyme activities in callus tissue of salt

tolerant and salt sensitive cultivars (Gossypium hirsu-tum L.). Plant Cell Rep. 13:498–503.

Hasegawa PM, Bressan R, Zhu JK, Bohnert H. 2000.Plant cellular and molecular responses to high salinity.Annu Rev Plant Physiol Plant Mol Biol. 51:463–499.

Hegedus A, Erdei S, Horvath G. 2001. Comparativestudies of H2O2 detoxifying enzymes in green andgreening barley seedlings under cadmium stress. PlantSci. 160:1085–1093.

Huang C, He W, Guo J, Chang X, Su P, Zhang L.2005. Increased sensitivity to salt stress in an ascor-bate-deficient Arabidopsis mutant. J Exp Bot.56:3041–3049.

Kristal AR, Lampe JW. 2002. Brassica Vegetables andprostate cancer risk: a review of the epidemiologicalevidence. Nutr Cancer. 42:1–9.

Lichtenthaler HK, Buschmann C. 2001. Chlorophylls andcarotenoids: measurement and characterization byUV–vis spectroscopy. In: Wrolstad RE, Acree TE,An H, Decker EA, Penner MH, Reid DS, SchwartzSJ, Shoemaker CF, Sporns P, editors. Current proto-cols in food analytical chemistry. New York NY: JohnWiley and Sons; p. F4.3.1–4.3.8.

Lin CC, Kao CH. 2000. Effect of NaCl stress on H2O2metabolism in rice leaves. Plant Growth Regul.30:151–155.

Manchanda G, Garg N. 2008. Salinity and its effects onthe functional biology of legumes. Acta Physiol Plant.30:595–618.

McKersie BD, Leshem YY. 1994. Stress and stress copingin cultivated plants. Netherlands: Kluwer AcademicPublishers; p. 15–78.

Miyake C, Asada K. 1992. Thylakoid-bound ascorbateperoxidase in spinach chloroplasts and photoreduc-tion of its primary oxidation product monodehydroas-corbate radicals in thylakoids. Plant Cell Physiol.33:541–553.

Nandwal AS, Godara M, Sheokand S, Kamboj DV,Kundu BS, Kuhad MS, Sharma SK. 2000. Salinityinduced changes in plant water status nodule func-tioning and ionic distribution in phenotypically differ-ing genotype of Vigna radiate L. J Plant Physiol.156:352–359.

Niyogi KK. 1999. Photoprotection revisited: genetic andmolecular approaches. Annu Rev Plant Physiol PlantMol Biol. 50:333–359.

Noctor G, Foyer CH. 1998. Ascorbate and glutathione:keeping active oxygen under control. Annu Rev PlantPhysiol Plant Mol Biol. 49:249–279.

Parida AK, Das AB. 2005. Salt tolerance and salinityeffects on plants: a review. Ecotox Environ Safe.60:324–349.

Pignocchi C, Foyer CH. 2003. Apoplastic ascorbatemetabolism and its role in the regulation of cellsignaling. Curr Opin Plant Biol. 6:379–389.

Premachandra GS, Saneoka H, Fujita K, Ogata S. 1992.Leaf water relations, osmotic adjustment, cell mem-brane stability, epi-cuticular wax load and growth asaffected by increasing water deficits in Sorghum. JExp Bot. 43:1569–1576.

SAS Institute Inc. 2004. SAS/STAT user’s guide, version9.1. Cary (NC): SAS Publishing.

Sekmen AH, Türkan I, Takio S. 2007. Differentialresponses of antioxidative enzymes and lipid perox-idation to salt stress in salt-tolerant Plantago maritime

10 A. Tayebi-Meigooni et al.

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014

and salt-sensitive Plantago media. Physiol Plantarum.131:399–411.

Shalata A, Neumann PM. 2001. Exogenous ascorbic acid(vitamin C) increases resistance to salt stress andreduces lipid peroxidation. J Exp Bot. 52:2207–2211.

Shalata A, Tal M. 1998. The effect of salt stress on lipidperoxidation and antioxidants in the leaf of the culti-vated tomato and its wild salt tolerant relative Lyco-persicon pennellii. Physiol Plantarum. 104:169–174.

Sinha S, Bhatt K, Pandey K, Singh S, Saxen R. 2003.Interactive metal accumulation and its toxic effects un-der repeated exposure in submerged plant Najas indicaCham. Bull Environ Contam Toxicol. 70:696–704.

Smart RE, Bingham GE. 1974. Rapid estimates of relativewater content. Plant Physiol. 53:258–260.

Takamiya KI, Tsuchiya T, Ohta H. 2000. Degradationpathway(s) of chlorophyll: what has gene cloningrevealed? Trends Plant Sci. 5:426–431.

Tayebimeigooni A, Awang Y, Mahmood M, Selamat A,Wahab Z. 2012. Leaf water status, proline content,lipid peroxidation and accumulation of hydrogenperoxide in salinized Chinese kale (Brassica albogla-bra). J Food Agr Environ. 10:371–374.

Thompson JE, Ledge RL, Barber RF. 1987. The role offree radicals in senescence and wounding. NewPhytol. 105:317–344.

Webb RP, Allen RD. 1995. Isolation and characterizationof a cDNA for spinach cytosolic ascorbate peroxidase.Plant Physiol. 108:13–25.

Zhang S, Weng J, Pan J, Tu T, Yao S, Xu C. 2003. Studyon the photo-generation of superoxide radicals inPhotosystem II with EPR spin trapping techniques.Photosynth Res. 75:41–48.

Zhu JK. 2000. Genetic analysis of plant salt toleranceusing Arabidopsis. Plant Physiol. 124:941–948.

Acta Agriculturae Scandinavica, Section B - Soil & Plant Science 11

Dow

nloa

ded

by [

Dr

Ali

Gha

sem

zade

h] a

t 01:

00 0

2 Fe

brua

ry 2

014