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Scientia Horticulturae 148 (2012) 197–205

Contents lists available at SciVerse ScienceDirect

Scientia Horticulturae

journa l h o me page: www.elsev ier .com/ locate /sc ihor t i

lleviation of salt-induced adverse effects in pepper seedlings by seedpplication of glycinebetaine

hmet Korkmaza,b,∗, Rauf S irikc i a, Ferit Kocac ınarc, Özlem Degera, Ali Rıza Demirkırıand

Kahramanmaras Sutcu Imam University, Faculty of Agriculture, Department of Horticulture, Kahramanmaras 46060, TurkeyKahramanmaras Sutcu Imam University, Research and Development Center For University – Industry and Public Relations, Kahramanmaras 46060, TurkeyKahramanmaras Sutcu Imam University, Faculty of Forestry, Department of Forest Engineering, Kahramanmaras 46060, TurkeyBingöl University, Faculty of Agriculture, Department of Soil Science and Plant Nutrition, Bingöl 12000, Turkey

r t i c l e i n f o

rticle history:eceived 17 May 2012eceived in revised form 5 September 2012ccepted 27 September 2012

eywords:apsicum annuumalondialdehyde

hotosynthesisalt stress

a b s t r a c t

In this study, the possibility of enhancing salt stress tolerance of pepper (Capsicum annuum L.) duringearly growth stages by seed application of glycinebetaine (GB) was investigated. To improve salt toleranceduring seedling stage, GB was applied in four different concentrations (0, 1, 5, or 25 mM) as a pre-sowingseed treatment. When the seedlings reached four fully developed true leaf stage, they were either exposedto salt stress (150 mM NaCl) for 2 weeks or allowed to grow under optimum conditions (0 mM NaCl). Saltstress applied through the root medium markedly suppressed plant growth, however, seed application ofGB provided significant protection against salt stress compared to non-GB-treated seedlings, considerablyenhancing photosynthesis and chlorophyll and proline contents of the seedlings. GB pre-treatment alsoimproved leaf water potential, relative water content, and superoxide dismutase (SOD) enzyme activity

uperoxide dismutase while reducing membrane permeability and lipid peroxidation. Moreover, GB pre-treatment reducedthe accumulation of Na+ and Cl− contents and prevented salt-induced K+ leakage thereby maintaining alower Na+/K+ ratio. Among the GB concentrations applied, the highest salt tolerance was obtained with5 mM GB pre-treatment. Results, therefore, indicate that GB, applied as pre-sowing seed treatment, couldbe used effectively to protect pepper seedlings from damaging effects of salt stress without any adverseeffect on seedling growth.

. Introduction

Soil salinity is a major constraint to agricultural productionecause it limits crop yield and restricts use of land previouslyncultivated. Salinity affects 19.5% of all irrigated land and 2.1%f dry-land agriculture worldwide (FAO, 2005). Each year there is aeterioration of 2 million ha (about 1%) of world agricultural landso salinity, leading to reduced or no crop productivity (Szabolcs,994). In addition to natural causes such as salty raining watersear and around the coasts and weathering of native rocks, low

recipitation and high surface evaporation and poor growing prac-ices have also aggravated increasing concentration of salts in thehizosphere (Mahajan and Tuteja, 2005). Secondary salinization,

Abbreviations: A, photosynthetic rate; E, transpiration; EC, electrical conduc-ivity; GB, glycinebetaine; gs , stomatal conductance; MDA, malondialdehyde; ROS,eactive oxygen species; RWC, relative water content; SOD, superoxide dismutase.∗ Corresponding author at: Kahramanmaras Sutcu Imam University, Faculty ofgriculture, Department of Horticulture, Kahramanmaras 46060, Turkey.el.: +90 344 280 2035; fax: +90 344 280 2002.

E-mail address: akorkmaz@ksu.edu.tr (A. Korkmaz).

304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.scienta.2012.09.029

© 2012 Elsevier B.V. All rights reserved.

in particular, exacerbates the problem where once productive agri-cultural lands are becoming unsuitable to cultivation due to poorquality irrigation water (Ashraf and Foolad, 2007).

Plants have evolved various mechanisms that allow them to tol-erate unfavorable environments for continued survival and growth(Sakamoto and Murata, 2000). One such mechanism ubiquitous toplants is the accumulation of certain low molecular weight organicmetabolites that are known collectively as compatible solutes orosmoprotectants (Bohnert et al., 1995). Generally, they protectplants from stress through different means, including contributionto cellular osmotic adjustment, detoxification of reactive oxygenspecies, protection of cellular membrane integrity, and stabilizationof enzymes/proteins (Yancey et al., 1982; Bohnert and Jensen, 1996;Ashraf and Foolad, 2007). These solutes include proline, sucrose,polyols, trehalose and quaternary ammonium compounds (QACs)such as choline and various betaines (Rhodes and Hanson, 1993).

Among the QACs, glycinebetaine (GB) occurs most abundantlyin response to osmotic stress in plants (Ashraf and Foolad, 2007).

The molecular features of GB make it possible to interact with bothhydrophobic and hydrophilic domains of macromolecules withoutdisrupting cellular functions (Sakamoto and Murata, 2002). In addi-tion to its vital role in maintenance of turgor pressure, GB has also

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een shown to protect functional proteins, enzymes (e.g. Rubisco),nd lipids of the photosynthetic apparatus and to maintain elec-ron flow across the thylakoid membranes (Xing and Rajashekar,999; Allakhverdiev et al., 2003; Hussain et al., 2008). GB is knowno accumulate in response to abiotic stress in several crop species,ncluding sugar beet, wheat, spinach, barley, and sorghum (Ashrafnd Foolad, 2007; Chen and Murata, 2008). In these species, tol-rant genotypes normally accumulate GB in higher amounts thanensitive genotypes in response to stress (Rhodes and Hanson,993). In plants that do not accumulate GB, exogenous applica-ions elevate tolerance to various abiotic stresses, improving plantrowth and survival (Park et al., 2006; Farooq et al., 2008). Pep-er, being one of the most important and widespread crops in theorld, is classified as moderately salt sensitive (De Pascale et al.,

003) and the sensitivity is especially high during seed germina-ion and early seedling stages (Chartzoulakis and Klapaki, 2000). Inur ongoing research, we previously established that pre-sowingeed treatment with GB enhanced germination and emergence per-ormance of pepper seeds under salt stress conditions (Korkmaznd S irikc i, 2011). However, to the best of our knowledge, veryittle information is available regarding the effects of GB on salttress tolerance of pepper seedlings. Among the very few, Khafagyt al. (2009) reported that exogenous application of GB partiallyounteracted the detrimental effects of salinity stress by mitigat-ng the harmful effects of salinity on thickness of the midrib regionnd mesophyll tissue of the leaf blade. Therefore, the aims of thisxperiment were to test the possibility that exogenous applicationf GB through pre-sowing seed treatment would protect peppereedlings from damaging effects of salt stress and to fully elu-idate the mechanism by which GB increases tolerance to salttress.

. Materials and methods

.1. Plant material, GB treatments and salt stress imposition

Seeds of ‘Sena’ red pepper (Capsicum annuum L.), all from theame seed lot, were obtained from Agricultural Research Insti-ute, Kahramanmaras, Turkey. Seeds were disinfested in 1% (activengredient) sodium hypochlorite solution for 10 min to eliminateossible seed-borne microorganisms, rinsed for 1 min under run-ing water then were dried for 30 min at room temperature.

A single layer of pepper seeds was placed in covered transpar-nt polystyrene boxes (10 cm × 10 cm × 4 cm) on double layers oflter paper wetted with 15 mL of 0 (control), 1, 5, or 25 mM GBSigma–Aldrich, St. Louis, MO, USA) solution. The boxes were kept at0 ◦C in the dark for 24 h. After GB application, seeds were rinsed for

min under running water and left to dry on paper towels for 24 hnder room conditions (20–22 ◦C and 50–60% relative humidity).ntreated dry seeds were also included as dry control.

The seeds were planted at a depth of 1.0 cm into 5.5 cm-deepat cells (75 cm3) filled with growth medium consisting of peat anderlite in the ratio of 3:1. The flats were watered regularly with tapater and kept in a growth chamber at 25 ± 1 ◦C (day/night) under

ool fluorescent lamps (100 �mol m−2 s−1) for 16 h day−1 photope-iod.

When the seedlings had fully developed 4 true leaves (42–45ays after planting), salt stress was initiated. Half of the seedlingsere watered with distilled water containing 25 mM NaCl, and

he NaCl concentration was increased to a final concentration of50 mM with daily increments of 25 mM to avoid salinity shock,

hile the other half of the plants were continued to be wateredith only distilled water. Salt stress lasted for 15 days, after which

he plants were assessed to determine the effects of salt stress andB treatments. The treatments were replicated four times with

lturae 148 (2012) 197–205

12 plants in each replication and all treatments were arranged in arandomized complete block design.

2.2. Determination of shoot fresh weight, leaf area, andchlorophyll content

All plants were cut at the medium surface and their freshweights were immediately recorded. Leaf area of all plants wasdetermined with leaf area meter (LiCor, Model LI-3100C, Lincoln,NE, USA).

Chlorophyll content was determined by taking fresh leaf sam-ples (0.5 g) from randomly selected three plants per each replicate.The samples were homogenized with 5 mL of acetone (80%,v/v) using pestle and mortar and filtered through a filter paper(Whatman, No. 2). The absorbance was measured with UV/visiblespectrophotometer (Optima SP3000-Plus, Tokyo, Japan) at 663and 645 nm and chlorophyll contents were calculated using theequations proposed by Lichtenthaler (1987) given below. Totalchlorophyll content was expressed as Chl a + Chl b.

Chl a (mg/g FW) = 11.75 × A663 − 2.35 × A645

Chl b (mg/g FW) = 18.61 × A645 − 3.96 × A663

2.3. Gas exchange measurements

Measurements of gas exchange attributes such as photosyn-thetic rate (A), transpiration (E), and stomatal conductance (gs)were performed on a fully expanded true leaf (from top) on twoplants from each replicate using a portable gas exchange system(Model: GFS3000, Walz, Effeltrich, Germany). Photosynthetic pho-ton flux density (supplied by red/blue light-emitting diode source)during the measurements was maintained at 100 �mol m−2 s−1

which was the same light intensity the plants were exposed to inthe growth chamber. Leaf temperature was maintained at 25 ◦C,and chamber CO2 concentration of 380 ppm was supplied by a CO2injector. Each leaf was allowed to reach a steady state of CO2 uptakebefore the measurements were taken.

2.4. Membrane permeability, relative water content and waterpotential measurements

In order to assess membrane permeability, electrolyte leakagewas determined according to Korkmaz et al. (2007). Leaf discs (1 cmin diameter) from randomly chosen two plants per replicate weretaken from the middle portion of fully developed youngest leaf andwashed with distilled water to remove surface contamination. Thediscs were placed in individual stoppered vials containing 20 mLof distilled water. After incubating the samples at room tempera-ture on a shaker (150 rpm) for 24 h, the electrical conductivity (EC)of the bathing solution (EC1) was determined. The same sampleswere then placed in an autoclave at 121 ◦C for 20 min and a secondreading (EC2) was determined after cooling the solution to roomtemperature. The electrolyte leakage was calculated as EC1/EC2 andexpressed as percentage.

To determine relative water content (RWC), leaf discs (1 cm indiameter) from randomly chosen two plants per replicate takenfrom the middle portion of fully developed third leaf (to exclude

the age effect) were used. Discs were weighed (fresh wt, FW) andthen immediately floated on distilled water in a Petri dish for 5 hin the dark. Turgid weights (TW) of leaf discs were obtained afterdrying excess surface water with paper towels. Dry weights (DW)

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f discs were measured after drying at 75 ◦C for 48 h. RWC wasalculated using the following formula:

WC(%) =[

FW − DWTW − DW

]× 100

eedling leaf water potential was determined by using a pressurehamber (PMS Inst., Model 1000, Albany, OR, USA).

.5. Determination of proline and MDA contents and SOD enzymectivity

Proline content was determined according to the methodescribed by Bates et al. (1973). Fresh leaf material (0.5 g) wasomogenized in 10 mL of 3% aqueous sulfosalicylic acid and filteredhrough Whatman’s No. 2 filter paper. Half milliliter of the filtrateas mixed with 1 mL of acid-ninhydrin and 1 mL of glacial acetic

cid in a test tube. The mixture was placed in a water bath for 1 ht 100 ◦C. The reaction mixture was extracted with 4 mL toluenend the chromophore containing toluene was aspirated, cooled tooom temperature, and the absorbance was measured at 520 nmith UV/visible spectrophotometer. Appropriate proline standardsere included for the calculation of proline in the samples.

The MDA concentration was determined according to theethod of Zhang et al. (2005) with some modification. Fresh shoot

amples (0.25 g) were homogenized in 3 mL of 10% trichloroaceticcid and centrifuged at 10,000 × g for 15 min. The supernatant wasollected and 1 mL was mixed with 1 mL of 0.6% thiobarbituric acid.he mixture was placed in a water bath set at 100 ◦C for 20 min,ooled quickly, and centrifuged at 10,000 × g for 10 min after whichts absorbance was determined at 532, 600, and 450 nm. The MDAoncentration was calculated according to the following formula:

DA (�mol g−1 FW) = 6.45 × (A532 − A600) − 0.56 × A450

The activity of SOD was determined using the slightly modi-ed method of Xu et al. (2008). Fresh leaf samples (0.5 g) wereapidly extracted in a pre-chilled mortar on an ice bath with 5 mL ofce-cold 100 mM phosphate buffer (pH 7.8) containing 1 mM EDTAnd 5% (w/v) PVP. After centrifugation at 10,000 × g for 30 mint 4 ◦C, the supernatant was used for SOD (EC 1.15.1.1) analysis.ne hundred �L of the enzyme extract was mixed with 2.465 mLf 100 mM phosphate buffer (pH 7.8), 75 �L of 55 mM methio-ine, 300 �l of 0.75 mM nitroblue tetrazolium (NBT) and 60 �Lf 0.1 mM riboflavin in a test tube. The test tubes containing theeaction solution were irradiated under 2 fluorescent light tubes40 �mol m−2 s−1) for 10 min. The absorbance measured at 560 nmith UV/visible spectrophotometer. Blanks and controls were run

n the same manner but without illumination and enzyme, respec-ively. One unit of SOD activity was defined as the amount ofnzyme that would inhibit 50% of NBT photo reduction.

.6. Determination of Na+, K+ and Cl− contents

Before determining Na+, K+ and Cl− contents of peppereedlings, dried shoots of all plants from each replicate were groundogether and a working sample was created. Na+ and K+ contentsere determined by atomic absorption spectrophotometry (Model

02, Perkin-Elmer, MA, USA) after wet digestion of dried tissues in a:1 nitric: perchloric acid mixture. Determination of chloride wasarried out by colorimetric titration with AgNO3 after extractionith water (Walinga et al., 1995).

.7. Statistical analysis

Data were subjected to analysis of variance (ANOVA) using thestat statistical software program and mean separation was per-

ormed by Fisher’s least significant difference (LSD) test if F test was

lturae 148 (2012) 197–205 199

significant at p = 0.05. Experiments were repeated twice and sincethere were no significant differences between the results of the twoexperiments, the data from both experiments were combined andthe mean values are presented (n = 8).

3. Results

Salt stress applied through the root medium markedly sup-pressed plant growth, reducing plant mass (Fig. 1a), leaf area(Fig. 1b) and leaf chlorophyll content (Fig. 1c). Plants subjected tosalt stress were half the size of those grown under optimum con-ditions and had slightly lowered chlorophyll content. Pre-sowingseed treatment with increasing concentrations of GB positivelyaffected plant growth under optimum conditions causing signif-icant enhancements in chlorophyll content and leaf area. Plantstreated with 25 mM GB had greater leaf area than plants emergingfrom both dry seeds and seeds treated with 0 mM GB. Simi-larly, plants pre-treated with 5 mM GB had higher leaf chlorophyllcontent compared to both control treatments (Fig. 1c). Addition-ally, under salt stress conditions, seed treatment with increasingconcentrations of GB had also positive influence on the growthof pepper seedlings (Fig. 1a–c). Even though all GB treatmentsimproved seedling mass, leaf area and chlorophyll content, treat-ing the seeds with 5 mM GB prior to sowing resulted in the heaviestseedlings, greatest leaf area and highest chlorophyll content.

Salt stress severely affected membrane integrity and tissuewater status, causing pronounced increases in electrolyte leakageand reductions in leaf water potential and RWC of pepper seedlings(Fig. 2a–c). Pre-treatment with GB did not improve seedling’s leafwater potential under salt stress conditions except that seedlingstreated with 5 mM GB exhibited slightly higher water potentialthan plants obtained from both dry seeds and seeds treated with0 mM GB (Fig. 2a). Membrane leakage and RWC of pepper seedlingswere positively affected by exogenous GB applications under salin-ity stress and plants pre-treated with 1 mM GB had the lowestmembrane leakage (Fig. 2b) while those treated with 5 mM GB hadthe highest RWC (Fig. 2c). In plants not exposed to salt stress condi-tions, however, GB applications had no apparent effect on seedlingwater potential and tissue membrane leakage (Fig. 2a and b). On theother hand, plants pre-treated with 1 and 5 mM GB had increasedtissue RWC compared to the two control treatments (Fig. 2c).

Salt stress suppressed all photosynthetic parameters, resultingin significant reductions in A (Fig. 3a), gs (Fig. 3b), and E (Fig. 3c).Seed treatment with increasing concentrations of GB did not havea notable effect on photosynthetic parameters of plants grownunder optimum conditions but it considerably improved the pho-tosynthetic capacity of salt-stressed plants (Fig. 3a–c). Even thoughimprovements in gs and E were not highly pronounced, A, on theother hand, was increased by GB applications up to 50% comparedto non-GB-applied seedlings, and the highest A was measured onseedlings treated with 5 mM GB (Fig. 3a).

Proline concentration in the leaves of pepper seedlingsincreased markedly in response to salt stress (Fig. 4a). Althoughseed application of GB did not result in any significant increasein proline content of seedlings grown under optimum conditions,GB pre-treatment caused up to 15% increase in proline content ofseedlings exposed to salt stress. Seed application of GB reducedlipid peroxidation (MDA content) in pepper seedlings exposed tosalt stress and the most reduction in MDA content was observedin 5 mM and 25 mM GB treated plants (Fig. 4b). MDA contents ofseedlings not exposed to salinity stress, however, were not influ-

enced by GB applications except that 5 mM GB-treated seedlingshad slightly higher MDA content compared to the two controltreatments. Moreover, pre-sowing seed treatment with increasingconcentrations of GB boosted SOD enzyme activity under saline

200 A. Korkmaz et al. / Scientia Horticulturae 148 (2012) 197–205

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ig. 1. Effect of pre-sowing seed treatment with GB on pepper seedling shoot fresaline (150 mM NaCl) conditions. Vertical bars represent mean ± SE (n = 8).

onditions (Fig. 4c). Seedlings obtained from 5 mM and 25 mM GBreatments exhibited higher enzyme activity than those obtainedrom non-GB-treated seeds. Under optimum conditions, however,re-treatment with increasing concentrations of GB caused onlylight increase in the SOD enzyme activity.

Na+ and Cl− accumulation in the leaves were increased by 4–5-old and by 2–3-fold, respectively, when the plants were exposedalt stress (Fig. 5a and b). Pre-sowing seed treatment with increas-ng concentrations of GB positively affected Na+ and Cl− contentsf seedlings causing reductions, and seedlings pre-treated with

mM GB prior to sowing had the lowest Na+ and Cl− contents.ven though, leaf K+ content was not increased by GB applicationsFig. 5c), Na+/K+ ratio was considerably reduced by GB treatmentsn salt-stressed plants (Fig. 5d).

. Discussion

Growth of pepper seedlings was found to be adversely affectedy salt stress and salinity-induced impairment in growth has

ght (a), leaf area (b), and chlorophyll content (c) under optimum (0 mM NaCl) and

already been reported previously in a number of crops such aseggplant (Abbas et al., 2010), tomato (Chen et al., 2009), soybean(Essa, 2002) and rice (Lutts, 2000). Data presented in this study, onthe other hand, indicated that GB application through seed soakingprotected pepper seedlings to a certain extent from the damagingeffects of salt stress. Plants pre-treated with GB were consider-ably larger in size as indicated by higher seedling mass (Fig. 1a)and leaf area (Fig. 1b) and had significantly higher chlorophyll con-tent (Fig. 1c) than those that were not pre-treated with GB. Theseresults correlate well with the findings of Yang and Lu (2005) whoreported that root application of 10 mM GB resulted in higher plantmass when maize seedlings were subjected to 100 mM NaCl stress.They also reported that GB-treated plants exhibited significantlyhigher chlorophyll and carotenoid contents than non-GB-treatedplants. Similar results were also reported by Meloni and Martinez

(2009) who found that exogenously applied GB in the rate of 8 mMincreased the dry weight of salt-stressed vinal (Prosopis ruscifoliaGriesbach) seedlings by 16% in comparison to seedlings treatedwith NaCl stress alone.

A. Korkmaz et al. / Scientia Horticulturae 148 (2012) 197–205 201

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ig. 2. Effect of pre-sowing seed treatment with GB on pepper seedling leaf water pnder optimum (0 mM NaCl) and saline (150 mM NaCl) conditions. Vertical bars rep

Water potential of cells or plant tissues is adversely affectedhen plants are exposed to various types of osmotic stress includ-

ng salt stress. GB has long been considered as vital compatiblesmotic solute that accumulates in tissues of plants under stressnd protects them from osmotic stress through osmotic adjust-ent (Ashraf and Foolad, 2007). Exogenous application of GB has

een reported to improve water potential of maize plants underalt stress (Nawaz and Ashraf, 2007) and RWC of tobacco seedlingsnder drought stress (Ma et al., 2007). In accordance with thesereviously reported results, the results of the current study alsoevealed that seed treatment with 5 mM GB significantly improvedeaf water potential and RWC of pepper seedlings especially underalt stress conditions (Fig. 2a and c).

Cell membrane stability estimated by membrane leakage haseen widely used to differentiate stress tolerant genotypes from

tress susceptible ones in crop species and measuring electrolyteeakage reflects the extent of cell membrane injury (Rehman et al.,004; Lv et al., 2007). A dehydration stress caused by drought,alt or extreme temperatures can lead to the disruption of cellular

ial (a), membrane permeability (EC1/EC2) (b), and relative water content (RWC) (c)t mean ± SE (n = 8).

membranes and increased permeability due to increased solu-bilization and peroxidation of phospholipids. In this study, weobserved that salt stress caused marked increase in membranepermeability (Fig. 2b). However, plants pre-treated with 5 mMGB exhibited considerably lower electrolyte leakage than non-GB-treated seedlings (Fig. 2b), which confirms the findings of otherresearchers. For example, Liang et al. (2009) reported that mem-brane permeability in transgenic wheat lines generated by theintroduction of the BADH gene that causes over production of GBwas significantly lower than wild type plants when exposed to saltstress. Similarly, it was also reported that exogenous applicationof GB reduced the membrane leakage in tomato seedlings underchilling stress (Park et al., 2006) and in rice seedlings exposed tosalt stress (Demiral and Türkan, 2004) compared to control plants.

Osmotic stress conditions such as salinity or drought reduce the

gas exchange attributes such as A, gs, and E and the reduction in A inplants under stress can be attributed to a reduction in chlorophyllcontent as well as to the stomatal limitations (Delfine et al., 1999).We found in the present study that salt stress caused considerable

202 A. Korkmaz et al. / Scientia Horticulturae 148 (2012) 197–205

F stomao t mean

rmof(htsmgsptiagwec

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ig. 3. Effect of pre-sowing seed treatment with GB on photosynthetic rate (A) (a),

ptimum (0 mM NaCl) and saline (150 mM NaCl) conditions. Vertical bars represen

eduction in A as well as gs and E; however, pre-sowing seed treat-ent with GB ameliorated the salt-induced inhibitory effect on A

f pepper seedlings (Fig. 3a). Improved A might have been resultedrom both improved gs (Fig. 3b) and increased chlorophyll contentFig. 1c). Even though the effect of GB application on gs was notighly pronounced, chlorophyll content is significantly boosted byhe GB applications. Increased growth of pepper seedlings underalt stress as demonstrated by higher seedling mass and leaf areaay be associated with increased A, since GB application miti-

ated the decrease in photosynthetic capacity. Many studies havehown that GB application or overproduction of GB improved thehotosynthetic capacity of crop plants under various stress condi-ions. For example, foliar application of GB in the rate of 50 mMncreased A of drought and salt-stressed tomato and turnip plantsnd the improvement in A was reported to be due to increaseds (Mäkelä et al., 1999). In addition, by introducing betA gene intoheat, the GB-overaccumulating transgenic lines had elevated lev-

ls of chlorophyll content and exhibited higher levels of gs and Aompared to wild type plants under salt stress (He et al., 2010).

While there was no visible effect of GB application on prolineontent of seedlings grown under optimum conditions, seedlings

tal conductance (gs) (b), and transpiration (E) (c) of pepper seedlings grown under ± SE (n = 8).

treated with GB prior to sowing had notably higher proline contentcompared to non-GB-applied seedlings under salt stress conditions(Fig. 4a). Exogenously applied GB can be readily taken up by seeds orplants and be transported to other organs, where it can contributeto enhanced stress tolerance by boosting the ability to osmoticallyadjustment of plant cell, maintaining stabilization and integrityof cell membranes. Conflicting reports have been reported on theeffect of exogenous application of GB on proline content of plantsunder stress. Several studies have reported that application of GBinduced the accumulation of other osmotic substances such as sol-uble sugars and free proline in plant tissues under stress (Ashrafand Foolad, 2007; Chaum and Kirdmanee, 2010). Boosted prolinecontent may be attributed to the protective role of GB on some keyenzymes that catalyze the biosynthesis of soluble sugars and freeproline (Liang et al., 2009). On the contrary, Heuer (2003) and Abbaset al. (2010) reported that exogenous application of GB to tomatoand eggplant seedlings, respectively, grown under salt stress did

not alter the proline content.

Salt stress usually causes extensive damage to the cellular mem-branes of plants and some of the effects exerted by stress arecaused by reactive oxygen species (ROS) which are detrimental

A. Korkmaz et al. / Scientia Horticulturae 148 (2012) 197–205 203

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ig. 4. Effect of pre-sowing seed treatment with GB on proline content (a), MDA coaCl) and saline (150 mM NaCl) conditions. Vertical bars represent mean ± SE (n = 8

o all biological systems (Foyer et al., 1997). The decompositionf polyunsaturated fatty acids in the membranes by peroxida-ion generates MDA, a toxic byproduct. A remarkable increasen lipid peroxidation level in leaves of pepper seedlings underalinity and its decrease with GB pre-treatment suggest that exoge-ous GB application can largely protect the seedlings from salttress-generated oxidative damage (Fig. 4b). These results correlateell with those reported by Demiral and Türkan (2004) and Meloni

nd Martinez (2009) who reported that GB pre-treatment reducedevels of lipid peroxidation in rice and vinal seedlings subjected toalinity stress.

Salt tolerance is generally correlated with a more efficientntioxidative system and an increase in the activity of stressnzymes such as superoxide dismutase (SOD), catalase and perox-dases in response to salinity stress has been reported (Srivastava,002; Meloni et al., 2003). Salt-induced enzyme activity indicates

specific role in coping with the stress (Gueta-Dahan et al., 1997)nd our findings suggest that seedlings pre-treated with GB exhib-ted enhanced SOD activity under salt stress condition (Fig. 4c), andhat lower MDA contents determined in these plants may be due

(b), and SOD enzyme activity (c) of pepper seedlings grown under optimum (0 mM

to enhanced SOD enzyme activity. Similar results were reportedby several studies in which exogenous application of GB has beenfound to stimulate the activities of antioxidative enzymes includ-ing SOD in vinal (Meloni and Martinez, 2009) and rice (Demiral andTürkan, 2004) seedlings under salt stress and tobacco seedlingsunder water deficit conditions (Ma et al., 2007). Moreover, itwas also reported that transgenic cotton plants genetically engi-neered to biosynthesize more GB exhibited significantly higher SODenzyme activity compared to non-transgenic (wild type) plantsunder salt stress (Zhang et al., 2011).

One way to characterize the salinity tolerance is the plant’scapacity for osmotic adjustment, which allows growth to continueunder saline conditions (Heuer, 2003). It is generally accepted thatthis process is mainly achieved by exclusion of Na+ and increasedabsorption of K+ to maintain a proper Na+/K+ ratio in the tis-sues. Salt-induced accumulation of Na+ and Cl− and decrease in

K+ content is often observed in crop species (Zhu, 2003). In ourinvestigation, accumulation of both Na+ and Cl− in the leavesof pepper seedlings was increased significantly with salt stress,while that of K+ decreased (Fig. 5a–c). However, application of

204 A. Korkmaz et al. / Scientia Horticulturae 148 (2012) 197–205

F ts (c),

s

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ig. 5. Effect of pre-sowing seed treatment with GB on Na+ (a), Cl− (b), K+ contenaline (150 mM NaCl) conditions. Vertical bars represent mean ± SE (n = 8).

B checked the accumulation of Na+ and Cl− while sustaininghat of K+ maintaining a lower Na+/K+ ratio (Fig. 5d). Consideringhe K+ retention observed in salt-tolerant crops, the ameliorat-ng effect of GB on salt-induced K+ efflux would have significant

and Na+/K+ ratio (d) of pepper seedlings grown under optimum (0 mM NaCl) and

consequences for the salt tolerance of the plant (Meloni andMartinez, 2009).

Overall, the result of the present study revealed that pre-treatment with GB through seed soaking was effective in inducing

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alt tolerance in pepper seedlings and among the GB concentra-ion tested, 5 mM was found to be the most effective concentrationnducing the highest level of tolerance to salinity. The growthmprovement due to GB application was found to be relatedo improved photosynthetic capacity and leaf water potential,educed lipid peroxidation-linked membrane deterioration andnhancement in SOD activity, a key enzyme of the ROS scaveng-ng system. GB pre-treatment also reduced the accumulation ofa+ and Cl− and prevented salt-induced K+ leakage thereby main-

aining a lower Na+/K+ ratio. Thus, GB applied as pre-sowing seedreatment could be used as an ameliorative agent for protectingepper seedlings against the detrimental effects of salt stress andur efforts are also currently underway to determine the effects ofB application on the yield potential of pepper plants under salttress.

cknowledgements

This work is a part of a project supported by a grant (project no:08 O 398) from The Scientific and Technical Research Council ofurkey (TUBITAK) and we are grateful for financial support.

eferences

bbas, A., Ashraf, M., Akram, N.A., 2010. Alleviation of salt-induced adverse effectsin eggplant (Solanum melongena L.) by glycinebetaine and sugarbeet extracts.Sci. Hortic. 125, 188–195.

llakhverdiev, I., Hayashi, H., Nishiyama, Y., Ivanov, A.G., Aliev, J.A., Klimov, V.V.,Murata, N., Carpentier, R., 2003. Glycinebetaine protects the D1/D2/Cytb559complex of photosystem II against photo-induced and heat-induced inactiva-tion. J. Plant Physiol. 160, 41–49.

shraf, M., Foolad, M.A., 2007. Improving plant abiotic-stress resistance by exoge-nous application of osmoprotectants glycine betaine and proline. Environ. Exp.Bot. 59, 206–216.

ates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline forwater-stress studies. Plant Soil 39, 205–207.

ohnert, H.J., Nelson, D.E., Jenson, R.G., 1995. Adaptations to environmental stresses.Plant Cell 7, 1099–1111.

ohnert, H.J., Jensen, R.G., 1996. Strategies for engineering water-stress tolerance inplants. Trends Biotechnol. 14, 89–97.

hartzoulakis, K., Klapaki, G., 2000. Response of two greenhouse pepper hybrids toNaCl salinity during different growth stages. Sci. Hortic. 86, 247–260.

haum, S., Kirdmanee, C., 2010. Effect of glycinebetaine on proline, water use, andphotosynthetic efficiencies, and growth of rice seedlings under salt stress. Turk.J. Agric. Forest. 34, 517–527.

hen, T.H.H., Murata, N., 2008. Glycinebetaine: an effective protectant against abioticstress in plants. Trends Plant Sci. 13, 499–505.

hen, S., Gollop, N., Heuer, B., 2009. Proteomic analysis of salt-stressed tomato(Solanum lycopersicum) seedlings: effect of genotype and exogenous applicationof glycinebetaine. J. Exp. Bot. 60, 2005–2019.

e Pascale, S., Ruggiero, C., Barbieri, G., Maggio, A., 2003. Physiological responses ofpepper to salinity and drought. J. Am. Soc. Hortic. Sci. 128, 48–54.

elfine, S., Alvino, A., Villani, M.C., Loreto, F., 1999. Restriction to carbondioxideconductance and photosynthesis in spinach leaves recovering from salt stress.Plant Physiol. 117, 293–302.

emiral, T., Türkan, I., 2004. Does exogenous glycinebetaine affect antioxidativesystem of rice seedlings under NaCl treatment? J. Plant Physiol. 161, 1089–1100.

ssa, T.A., 2002. Effect of salinity stress on growth and nutrient composition of threesoybean (Glycine max L, Merrill) cultivars. J. Agron. Crop Sci. 188, 86–93.

AO, 2005. Global network on integrated soil management for sustainable use of salteffected soils. Available in: http://www.fao.org/ag/AGL/agll/spush/intro.htm

arooq, M., Aziz, T., Hussain, M., Rehman, H., Jabran, K., Khan, M.B., 2008. Glycine-betaine improves chilling tolerance in hybrid maize. J. Agron. Crop Sci. 194,152–160.

oyer, C.H., Lopez-Delgado, H., Dat, J.E., Scott, I.M., 1997. Hydrogen peroxide

and glutathione-associated mechanisms of acclamatory stress tolerance andsignaling. Physiol. Plantarum 100, 241–254.

ueta-Dahan, Y., Yaniv, Z., Zilinskas, B.A., Ben-Hayyim, G., 1997. Salt and oxidativestress: similar and specific responses and their relation to salt tolerance in citrus.Planta 204, 460–469.

lturae 148 (2012) 197–205 205

He, C., Yang, A., Zhang, W., Gao, Q., Zhang, J., 2010. Improved salt tolerance of trans-genic wheat by introducing betA gene for glycine betaine synthesis. Plant CellTissue Organ Cult. 101, 65–78.

Heuer, B., 2003. Influence of exogenous application of proline and glycinebetaine ongrowth of salt-stressed tomato plants. Plant Sci. 165, 693–699.

Hussain, M., Malik, M.A., Farooq, M., Ashraf, M.Y., Cheema, M.A., 2008. Improvingdrought tolerance by exogenous application of glycinebetaine and salicylic acidin sunflower. J. Agron. Crop Sci. 194, 193–199.

Khafagy, M.A., Arafa, A.A., El-Banna, M.F., 2009. Glycinebetaine and ascorbic acid canalleviate the harmful effects of NaCl salinity in sweet pepper. Aust. J. Crop Sci. 3,257–267.

Korkmaz, A., Uzunlu, M., Demirkıran, A.R., 2007. Acetyl salicylic acid alleviateschilling-induced damage in muskmelon plants. Can. J. Plant Sci. 87, 581–585.

Korkmaz, A., S irikc i, R., 2011. Improving salinity tolerance of germinating seedsby exogenous application of glycinebetaine in pepper. Seed Sci. Technol. 39,377–388.

Liang, C., Zhang, X.Y., Luo, Y., Wang, G.P., Zouand, Q., Wang, W., 2009. Overaccumu-lation of glycinebetaine alleviates the negative effects of salt stress in wheat.Russ. J. Plant Physiol. 56, 370–376.

Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosyntheticbiomemranes. Methods Enzymol. 148, 350–382.

Lutts, S., 2000. Exogenous glycine betaine reduces sodium accumulation in salt-stressed rice plants. Int. Rice Res. Notes 25, 39–40.

Lv, S., Yang, A., Zhang, K., Wang, L., Zhang, L., 2007. Increase of glycinebetaine syn-thesis improves drought tolerance in cotton. Mol. Breed. 20, 233–248.

Ma, X.L., Wang, Y.J., Xie, S.L., Wang, C., Wang, W., 2007. Glycinebetaine applicationameliorates negative effects of drought stress in tobacco. Russ. J. Plant Physiol.54, 534–541.

Mahajan, S., Tuteja, N., 2005. Cold, salinity and drought stresses: an overview. Arch.Biochem. Biophys. 444, 139–158.

Mäkelä, P., Kontturib, M., Pehu, E., Somersalo, S., 1999. Photosynthetic responseof drought- and salt-stressed tomato and turnip rape plants to foliar-appliedglycinebetaine. Physiol. Plantarum 105, 45–50.

Meloni, D.A., Martinez, C.A., 2009. Glycinebetaine improves salt tolerance in vinal(Prosopis ruscifolia Griesbach) seedlings. Braz. J. Plant Physiol. 21, 233–241.

Meloni, D.A., Oliva, M.A., Martinez, C.A., Cambraia, J., 2003. Photosynthesis and activ-ity of superoxide dismutase, peroxidase and glutathione reductase in cottonunder salt stress. Environ. Exp. Bot. 49, 69–76.

Nawaz, K., Ashraf, M., 2007. Improvement in salt tolerance of maize by exoge-nous application of glycinebetaine: growth and water relations. Pak. J. Bot. 39,1647–1653.

Park, R.J., Jeknic, Z., Chen, T.H.H., 2006. Exogenous application of glycinebetaineincreases chilling tolerance in tomato plants. Plant Cell Physiol. 47, 706–714.

Rehman, H., Malik, S.A., Saleem, M., 2004. Heat tolerance of upland cotton duringfruiting stage evaluated using cellular membrane thermostability. Field CropsRes. 85, 149–158.

Rhodes, D., Hanson, A.D., 1993. Quaternary ammonium and tertiary sulfonium com-pounds in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 357–384.

Sakamoto, A., Murata, N., 2000. Genetic engineering of glycinebetaine synthesis inplants: current status and implication for enhancement of stress tolerance. J.Exp. Bot. 51, 81–88.

Sakamoto, A., Murata, N., 2002. The role of glycine betaine in the protection of plantsfrom stress: clues from transgenic plants. Plant Cell Environ. 25, 163–171.

Srivastava, L.M., 2002. Plant Growth and Development. Hormones and the Environ-ment. Academic Press, Oxford.

Szabolcs, I., 1994. Soil sand salinisation. In: Pessarakali, M. (Ed.), Handbook of Plantand Crop Stress. Marcel Dekker, New York, pp. 3–11.

Walinga, I., Van Der Lee, J.J., Houba, V.J.G., Van Vark, V., Novazamsky, I., 1995. PlantAnalysis Manual. Springer, Dordrecht, The Netherlands.

Xing, W., Rajashekar, C.B., 1999. Alleviation of water stress in beans by exogenousglycine betaine. Plant Sci. 148, 185–195.

Xu, P.L., Guo, Y.K., Bai, J.G., Shang, L., Wang, X.J., 2008. Effects of long-term chillingon ultrastructure and antioxidant activity in leaves of two cucumber cultivarsunder low light. Physiol. Plantarum 132, 467–478.

Yancey, P.H., Clark, M.E., Hand, S.C., Bowlus, R.D., Somero, G.N., 1982. Living withwater stress: evolution of osmolyte systems. Science 217, 1212–1222.

Yang, X., Lu, C., 2005. Photosynthesis is improved by exogenous glycinebetaine insalt-stressed maize plants. Physiol. Plantarum 124, 343–352.

Zhang, J.H., Huang, W.D., Liu, Y.P., Pan, Q.H., 2005. Effects of temperature acclimationpretreatment on the ultrastructure of mesophyll cells in young grape plants(Vitis vinifera L. cv, Jingxiu) under cross-temperature stresses. J. Int. Plant Biol.47, 959–970.

Zhang, K., Guo, N., Lian, L., Wang, J., Lv, S., Zhang, J., 2011. Improved salt toleranceand seed cotton yield in cotton (Gossypium hirsutum L.) by transformation withbetA gene for glycinebetaine synthesis. Euphytica, 1–16.

Zhu, J.K., 2003. Regulation of ion homeostasis under salt stress. Curr. Opin. Plant Biol.6, 441–445.