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Environmental and Experimental Botany 67 (2010) 495–501

Contents lists available at ScienceDirect

Environmental and Experimental Botany

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

nhancing chilling stress tolerance of pepper seedlings by exogenouspplication of 5-aminolevulinic acid

hmet Korkmaza,∗, Yakup Korkmaza, Ali Rıza Demirkıranb

Kahramanmaras Sutcu Imam University, Faculty of Agriculture, Department of Horticulture, Avsar Kampusu, Kahramanmaras 46060, TurkeyKahramanmaras Sutcu Imam University, Faculty of Agriculture, Department of Soil Science, Avsar Kampusu, Kahramanmaras 46060, Turkey

r t i c l e i n f o

rticle history:eceived 13 May 2009eceived in revised form 14 July 2009ccepted 19 July 2009

eywords:ntioxidant enzymesapsicum annuum

a b s t r a c t

In this study, the possibility of enhancing chilling stress tolerance of pepper (Capsicum annuum L.) dur-ing early growth stages by exogenous application of 5-aminolevulinic acid (ALA) was investigated. Toimprove chilling tolerance during seedling stage, ALA was applied in various concentrations (0, 1, 10, 25and 50 ppm) through three different methods (seed soaking, foliar spray, or soil drench). After ALA appli-cations, the plants were subjected to chilling stress at 3 ◦C for 2 days. Although all ALA application methodsimproved chilling stress tolerance in pepper seedlings, seed soaking and foliar spray provided better pro-tection against chilling stress compared to soil drench. Exogenous application of ALA provided significantprotection against chilling stress compared to non-ALA-treated seedlings, significantly enhancing plant

hilling stress tolerance

lectrolyte leakageas exchangelant growth

mass and chlorophyll, sucrose, and proline contents. ALA pre-treatment also increased relative watercontent, stomatal conductance and superoxide dismutase (SOD) enzyme activity and reduced membranepermeability. Of the ALA concentrations, the highest chilling tolerance was obtained with 25 ppm ALApre-treatment. Results indicate that ALA which is considered as an endogenous plant growth regulatorcould be used effectively to protect pepper seedlings from damaging effects of chilling stress without any

g grow

adverse effect on seedlin

. Introduction

The optimum growth temperature for pepper (Capsicumnnuum L.) is between 21 and 27 ◦C, with growth reduced below 12nd above 30 ◦C (Wien, 1997). Exposure of plants from tropical orubtropical origin to chilling temperatures may stunt plant growth,nduce wilting, cause necrotic lesions on leaves, and increase sus-eptibility to diseases and pathogens (Hällgreen and Öquist, 1990;orkmaz and Dufault, 2001). Plant growth may deteriorate as aonsequence of impaired photosynthesis and respiration and dis-upted water relations, membrane integrity, and hormonal balanceAllen and Ort, 2001; Korkmaz et al., 2007).

Pepper is grown extensively in western and southern Turkey,nd although climatic conditions in these areas are ideal for grow-ng pepper, increasing yield by multiple harvests requires field

lanting in early March before the last killing frost. For example, inhe Kahramanmaras province (one of the major commercial pepperroduction areas in Turkey), the mean air temperatures range from0 to 15 ◦C and the mean min temperatures fluctuate between 3 and

Abbreviations: ALA, 5-aminolevulinic acid; EC, electrical conductivity; NBT, nitrolue tetrazolium; RWC, relative water content; SOD, superoxide dismutase.∗ Corresponding author. Tel.: +90 344 219 1564; fax: +90 344 219 1526.

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

098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2009.07.009

th.© 2009 Elsevier B.V. All rights reserved.

7 ◦C on typical planting dates (Korkmaz, 2009). Once the seedlingshave been transplanted in the field, they may be exposed to tem-peratures cycling between chilling and optimum for weeks beforetemperatures finally stabilize. In some cases, late frosts occur whichkill the newly transplanted seedlings in the field or if not severeenough, stagnate early plant growth and field establishment.

It is known that 5-aminolevulinic acid (ALA) is a key precursorin the biosynthesis of all porphyrins compounds such as chloro-phyll, heme, and phytochrome (Wang et al., 2005). Exogenousapplications of ALA have been found to regulate plant growth anddevelopment and to enhance chlorophyll biosynthesis and photo-synthesis thus increasing crop yield (Hotta et al., 1997). Treatingrice, barley, potato and garlic plants at early growth stages withsuitable concentrations of ALA promoted plant growth and photo-synthetic rates resulting in significant yield enhancements (Tanakaet al., 1992). ALA applied at low concentrations is also known toenhance plant’s tolerance to cold (Hotta et al., 1998; Wang et al.,2004) and salinity stresses (Watanabe et al., 2000; Nishihara et al.,2003; Zhang et al., 2006) and exhibit herbicidal effects if used atconcentrations over 5 mM (Kumar et al., 1999), suggesting that

ALA has a great application potential in agricultural productionas a new non-toxic endogenous substance (Wang et al., 2003). Inour ongoing research, we previously established that pre-sowingseed treatment with 50 ppm ALA enhanced germination and emer-gence performance of pepper seeds under chilling stress conditions

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Korkmaz and Korkmaz, 2009). However, to our knowledge, no spe-ific information is available regarding the effects of ALA on chillingtress tolerance of pepper seedlings. Moreover, the mechanisms ofLA in promoting stress tolerance in plants also need to be fullylucidated. Therefore, this study provides the first evidence thatLA protected pepper seedlings against chilling stress. It also con-

ributes significantly to our understanding of the role of ALA inromoting chilling stress tolerance. Our specific objectives were1) to compare three ALA application methods and (2) to deter-

ine the optimum ALA concentration that would provide the bestrotection against chilling stress.

. Materials and methods

.1. Plant material, ALA treatments and chilling stress imposition

Seeds of ‘Sena’ red pepper, all from the same seed lot, werebtained from Agricultural Research Institute, Kahramanmaras,urkey. Seeds were disinfested in 1% (active ingredient) sodiumypochlorite solution for 10 min to eliminate possible seed-borneicroorganisms, rinsed for 1 min under running water then were

ried 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, 1, 10, 25 or 50 ppm ALA (Sigmaldrich, St. Louis, MO, USA) solution. The boxes were kept at 20 ◦C

n the dark for 24 h. After ALA application, seeds were rinsed formin under running water and left to dry on paper towels for 24 hnder room conditions (20–22 ◦C and 50–60% relative humidity).

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.A second batch of seeds was also imbibed in distilled water

nder the same conditions prior to sowing to obtain seedlings foroliar spray and soil drench applications of ALA, and these seedlingsere grown under the same conditions. When the seedlings had

ully developed 4 true leaves (30–34 days after planting), half of theeedlings were sprayed with 0, 1, 10, 25 or 50 ppm ALA solutionsntil both sides of the leaves were completely wet while the otheralf was soil drenched with 25 ml (enough to cause run off) of 0, 1,0, 25 or 50 ppm ALA solution. For foliar application, a few dropsf surfactant (Tween 20) were added to ALA solutions to increasedherence and paper towels were laid on the growth medium torevent ALA solution from entering the growth medium. Threeays after foliar spray and soil drench application of ALA, all plantsseed soaked, foliar sprayed, and soil drenched) were subjected tohilling stress at 3 ± 0.5 ◦C for 48 h under the same light regime asentioned above. All plants were watered 2 h prior to and after

he chilling stress and were assessed 72 h after the end of chillingtress to determine the extent of chilling injury. The treatmentsere replicated four times with 12 plants in each replication and all

reatments were arranged in a randomized complete block design.or comparison purposes, plants not exposed to chilling stress andrown in the growth chamber at 25 ◦C were used as the control.

.2. Determination of visual damage

All plants were visually examined to determine the extent ofhilling injury and classified using the following scale: none: no vis-

ble symptoms, slight: small necrotic areas on shoots but withoutrowth restrictions (<5% of leaf area necrotic), moderate: well-efined necrotic areas on shoots (5–25% of leaf area necrotic),evere: extensive necrotic areas and severe growth reductions26–50% of leaf area necrotic but plant still alive), and killed: entire

rimental Botany 67 (2010) 495–501

plant necrotic and collapsed. By assigning values of 1, 2, 3, 4, and 5,respectively to each group, the average injury for each treatmentwas calculated (Korkmaz et al., 2007).

2.3. Chlorophyll content determination

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 (What-man No. 2). The absorbance was measured with a UV/visiblespectrophotometer (Spectramax Plus 384, Molecular Devices, CA,USA) at 663 and 645 nm and chlorophyll contents were calculatedusing the equations proposed by Lichtenthaler (1987) given below:

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

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

2.4. Stomatal conductivity

The youngest fully expanded leaves of randomly selected threeplants per replicate were chosen for gas exchange measurements,and stomatal conductivity was measured using a portable porom-eter (Model: AP4, Delta-T Devices, Cambridge, UK). Light intensityduring the measurements was maintained at 100 �mol m−2 s−1

which was the same light intensity that the plants were exposed toin the growth chamber.

2.5. Electrolyte leakage

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 percent.

2.6. Relative water content

Leaf discs (1 cm in diameter) from randomly chosen two plantsper replicate were taken from the middle portion of fully developedthird leaf (to exclude the age effect). Discs were weighed (fresh wt,FW) and then immediately floated on distilled water in a petri dishfor 5 h in the dark. Turgid weights (TW) of leaf discs were obtainedafter drying excess surface water with paper towels. Dry weights(DW) of discs were measured after drying at 75 ◦C for 48 h. Relativewater content (RWC) was calculated using the following formula:

RWC =[

FW − DWTW − DW

]× 100

2.7. Proline content determination

Proline content was determined according to the methoddescribed by Bates et al. (1973). Fresh leaf material (0.5 g) washomogenized in 10 ml of 3% aqueous sulfosalicylic acid and filtered

d Experimental Botany 67 (2010) 495–501 497

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Table 1Effect of ALA applications through different methods on the degree of visualdamage symptoms of pepper seedlings subjected to chilling stress. Values aremeans ± SE (n = 8).

Treatments Visual damage indexa (1–5)

App. methodSeed soak 2.22 ± 0.14Foliar spray 1.98 ± 0.12Soil drench 2.57 ± 0.14

ALA Con. (ppm)0 3.12 ± 0.161 2.21 ± 0.1710 2.10 ± 0.1525 1.88 ± 0.1250 1.98 ± 0.20

Significanceb L*Q*r2 0.56

Unstressed controlc 1.1 ± 0.02

ANOVAApp. method (M) **ALA Con. (C) ***M × C NS

NS, *, **, ***, not significant, significant at P < 0.05, 0.01 or 0.001, respectively.a

A. Korkmaz et al. / Environmental an

hrough 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 h at00 ◦C. The reaction mixture was extracted with 4 ml toluene andhe chromophore containing toluene was aspirated, cooled to roomemperature, and the absorbance was measured at 520 nm with aV/visible spectrophotometer. Appropriate proline standards were

ncluded for the calculation of proline in the samples.

.8. Determination of SOD enzyme activity

Fresh leaf samples (0.5 g) were rapidly extracted in a pre-chilledortar on an ice bath with 5 ml of ice-cold 100 mM phosphate

uffer (pH 7.8) containing 1 mM EDTA and 5% (w/v) PVP. Afterentrifugation at 10,000 × g for 30 min at 4 ◦C, the supernatantas used for SOD (EC 1.15.1.1) analysis. The activity of SOD wasetermined using the slightly modified method of Xu et al. (2008).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 methion-ne, 300 �l of 0.75 mM nitroblue tetrazolium (NBT) and 60 �l of.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 a UV/visible spectrophotometer. Blanks and controls were

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

.9. Plant mass determination and chemical analysis

All plants were cut at the growth medium surface and shootnd root fresh and dry weights (dried at 105 ◦C for 24 h) were deter-ined. Before determining carbohydrate (sucrose) and K+ contents

f pepper seedlings, dried shoots of all plants from each repli-ate were ground together and a working sample was created.ucrose content was determined according to Anthrone method asescribed by Morris (1948). K+ content was determined by atomicbsorption spectrophotometry (Model 302, PerkinElmer, MA, USA)fter wet digestion of dried tissues in a 3:1 nitric: perchloric acidixture.

.10. Statistical analysis

Data were subjected to analysis of variance (ANOVA) using theSTATC statistical software program. Experiments were repeated

wice and since there were no significant differences between theesults of two experiments, data from both experiments were com-ined and the mean values are presented (n = 8). If there was aignificant correlation between ALA concentrations and variableseasured or determined, polynomial regression analysis was per-

ormed to indicate significant effects using Sigma Stat statisticaloftware.

. Results

.1. Visual damage symptoms

ALA, applied via three methods within the range of 1–50 ppm,as effective in reducing visual injury symptoms of pepper

eedlings subjected to chilling stress. The method of ALA appli-ation significantly affected the degree of visual injury symptoms

aused by chilling stress (Table 1). Among the ALA applicationethods, foliar spray resulted in the least visual damage symptoms

ollowed by seed soaking. On the other hand, plants soil drenchedith ALA solutions were affected the most by chilling stress as

ndicated by significantly higher injury rating values.

1 = no injury, 5 = plant killed.b Linear (L) or quadratic (Q).c Mean for unstressed control given for comparison purposes only and not

included in the statistical analysis.

After exposure to chilling stress for 2 days, plants not treatedwith ALA (0 ppm) exhibited typical chilling injury symptoms inmoderate to severe levels (>25% leaf area necrotic) while ALA-treated seedlings were slightly damaged (<5% leaf area necrotic).The non-ALA-treated seedlings had wilted leaves and lost a con-siderable portion of their foliage due to necrotic areas comparedto ALA-treated seedlings. A curvilinear relationship was observedbetween ALA concentrations and visual injury which revealed thateven though all ALA concentrations within the range of 1–50 ppmwere effective in reducing the visual damage, the most effectiveconcentration was 25 ppm, and that plants treated with 25 ppmALA exhibited the least visual injury symptoms. The visual appear-ance of 25 ppm ALA-treated plants was very similar to that ofunstressed control plants which remained at 25 ◦C and neverexposed to chilling temperatures.

3.2. Chlorophyll content

ALA applied in increasing concentrations prior to chilling stressenhanced chlorophyll content of the seedlings which was expectedsince ALA is a precursor in biosynthesis of such compounds aschlorophyll (Table 2). Plants treated with ALA through differentmethods had similar Chl a, Chl b and total chlorophyll contents,indicating that ALA application methods did not influence chloro-phyll content. On the other hand, treating pepper seedlings withincreasing concentrations of ALA caused significant enhancementsin chlorophyll content and the relationship between ALA con-centrations and chlorophyll content was curvilinear. This meantthat raising ALA concentration from 0 to 25 ppm resulted inimprovement in chlorophyll content, but a further increase in ALAconcentration to 50 ppm caused a slight reduction.

3.3. Electrolyte leakage, relative water content and stomatalconductance

ALA application method did not influence membrane per-meability or stomatal conductance of pepper seedlings whereastissue RWC was affected significantly by the method of application(Table 3). Plants treated with ALA via foliar spray had considerably

498 A. Korkmaz et al. / Environmental and Expe

Table 2Effect of ALA applications through different methods on chlorophyll a, chlorophyll band total chlorophyll content of pepper seedlings subjected to chilling stress. Valuesare means ± SE (n = 8).

Treatments Chl a(mg g−1 FW)

Chl b(mg g−1 FW)

Chl a + b(mg g−1 FW)

App. methodSeed soak 20.6 ± 1.6 18.2 ± 1.5 38.8 ± 2.8Foliar spray 19.4 ± 1.6 22.1 ± 2.3 41.5 ± 3.7Soil drench 19.5 ± 1.5 23.6 ± 2.1 43.1 ± 3.5

ALA Con. (ppm)0 17.2 ± 2.3 16.2 ± 2.7 33.4 ± 4.61 17.6 ± 1.9 21.5 ± 2.4 39.0 ± 4.110 19.6 ± 2.1 22.0 ± 2.5 41.6 ± 4.625 24.9 ± 1.3 27.6 ± 2.4 52.5 ± 3.350 19.8 ± 2.1 19.1 ± 2.5 39.0 ± 4.2

Significancea L*Q** L*Q* L*Q**r2 0.57 0.36 0.66

Unstressed controlb 18.8 ± 1.7 20.3 ± 2.2 39.1 ± 3.9

ANOVAApp. method (M) NS NS NSALA Con. (C) * ** *M × C NS NS NS

NS, *, **, not significant, significant at P < 0.05 or 0.01, respectively.

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igher RWC compared to plants treated with other two applicationethods. Moreover, the main effect of ALA concentration was sig-

ificant and plants treated with increasing concentrations of ALAad considerably higher tissue RWC and stomatal conductance and

ower membrane permeability. The lowest electrolyte leakage andhe highest RWC and stomatal conductance were obtained fromeedlings treated with 25 ppm ALA.

.4. Plant mass

ALA, applied via three methods, significantly affected shoot andoot mass of pepper seedlings subjected to chilling stress (Table 4).

able 3ffect of ALA applications through different methods on membrane permeabilityEC1/EC2), relative water content (RWC) and stomatal conductance (gs) of peppereedlings subjected to chilling stress. Values are means ± SE (n = 8).

Treatments EC1/EC2 (%) RWC (%) gs (mmol m−2 s−1)

App. methodSeed soak 50.8 ± 2.3 81.4 ± 2.0 27.9 ± 1.7Foliar spray 50.4 ± 2.1 88.7 ± 1.3 25.2 ± 1.4Soil drench 54.3 ± 2.0 81.0 ± 1.5 24.6 ± 1.2

ALA Con. (ppm)0 62.2 ± 2.4 76.0 ± 2.6 16.2 ± 0.71 56.7 ± 2.7 82.4 ± 2.2 22.0 ± 0.910 48.9 ± 2.6 86.6 ± 1.6 27.8 ± 1.425 43.5 ± 2.5 87.7 ± 1.8 34.1 ± 1.950 47.9 ± 2.1 85.7 ± 2.0 29.3 ± 1.5

Significancea L***Q*** L*Q** L***Q***r2 0.83 0.52 0.80

Unstressed controlb 26.0 ± 3.6 89.4 ± 1.2 50 ± 2.3

ANOVAApp. method (M) NS *** NSALA Con. (C) *** *** ***M × C NS NS NS

S, *, **, ***, not significant, significant at P< 0.05, 0.01 or 0.001, respectively.a Linear (L) or quadratic (Q).b Mean for unstressed control given for comparison purposes only and not

ncluded in the statistical analysis.

rimental Botany 67 (2010) 495–501

The method of ALA application had a significant effect on seedlingshoot fresh weight, and the seedlings obtained from seed soak-ing and foliar spray treatments were noticeably heavier than thoseobtained from soil drench treatments. On the other hand, eventhough shoot dry weight of seedlings soil drenched with ALA solu-tions was lower than that of plants treated with ALA through seedsoaking and foliar spray, the relationship between ALA applicationmethod and shoot dry weight was insignificant. Moreover, therewas a linear relationship between ALA concentrations and seedlingfresh and dry weights, indicating that plant’s above-ground massincreased in response to higher concentrations of ALA. However,shoot fresh and dry weights of ALA-treated plants were still lowerthan those of unstressed plants.

Additionally, the method of ALA application significantlyaffected root fresh and dry weight (Table 4). Plants treated with ALAvia seed soaking and foliar spray had significantly higher root freshand dry weight compared to soil drenched plants. On the contrary,even though plants treated with increasing concentrations of ALAhad greater root fresh and dry weight compared to plants treatedwith 0 ppm ALA, the relationship between ALA concentration andthese variables was insignificant.

3.5. Sucrose, proline and K+ contents

The main effect of ALA application method was insignificant anddid not affect plant’s sucrose and proline contents (Table 5). K+ con-tent of plants, however, differed significantly with the method ofALA application, and plants treated with ALA via foliar spray hadthe highest K+ content while those soil drenched had the lowestK+ content. The main effect of ALA concentration was importantand a curvilinear relationship was observed between proline con-tent and ALA concentration. Increasing ALA concentrations up to25 ppm increased proline content, but a further increase in ALAconcentration to 50 ppm resulted in slight reductions in this vari-able. Moreover, sucrose content of seedlings was also significantlyaffected by the main effect of ALA concentration, and the highestsucrose content was obtained from plants treated with 25 ppm ofALA while those treated with 0 ppm ALA had the lowest sucrosecontent. Even though plants treated with increasing concentra-tions of ALA had greater K+ content compared to plants treatedwith 0 ppm ALA, the relationship between ALA concentration andK+ content was insignificant. Furthermore, 0 ppm ALA-treatedplants had considerably lower K+ content than unstressed controlplants.

3.6. SOD enzyme activity

Even though plants soil drenched with ALA solutions exhib-ited slightly lesser SOD enzyme activity than plants treated withALA through other two methods, the difference was negligible andstatistically insignificant (Table 5). The relationship between ALAconcentrations and SOD enzyme activity was very similar to thosereported above for other variables. Plants treated with increas-ing concentrations of ALA had clearly higher enzyme activity thanplants treated with 0 ppm ALA which had similar enzyme activityas unstressed control plants. The highest SOD enzyme activity wasobserved in plants treated with 25 ppm ALA.

4. Discussion

Data presented in this study indicated that ALA application

through seed soaking, foliar spray, or soil drench protected pep-per seedlings from the damaging effects of chilling stress. To thebest of our knowledge, these results are the first evidence that ALAhas protective effects against chilling stress in pepper seedlings.Plants pre-treated with ALA exhibited slight injury symptoms and

A. Korkmaz et al. / Environmental and Experimental Botany 67 (2010) 495–501 499

Table 4Effect of ALA applications through different methods on shoot and root fresh and dry weights of pepper seedlings subjected to chilling stress. Values are means ± SE (n = 8).

Treatments Shoot fresh wt (mg/plant) Shoot dry wt (mg/plant) Root fresh wt (mg/plant) Root dry wt (mg/plant)

App. methodSeed soak 1077 ± 59 93 ± 3 548 ± 24 39 ± 2Foliar spray 1171 ± 55 92 ± 4 544 ± 25 41 ± 2Soil drench 1015 ± 53 86 ± 4 437 ± 20 29 ± 1

ALA Con. (ppm)0 782 ± 45 73 ± 3 426 ± 24 31 ± 21 1150 ± 64 92 ± 4 528 ± 36 38 ± 310 1088 ± 59 89 ± 4 527 ± 28 36 ± 225 1266 ± 72 100 ± 5 539 ± 28 38 ± 250 1152 ± 82 98 ± 5 529 ± 37 39 ± 3

Significancea L*** L** NS NSr2 0.63 0.48 - -

Unstressed controlb 1390 ± 76 113 ± 7 551 ± 45 47 ± 6

ANOVAApp. method (M) * NS *** ***ALA Con. (C) *** *** * NSM × C NS NS NS NS

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S, *, **, ***, not significant, significant at P < 0.05, 0.01 or 0.001, respectively.a Linear (L) or quadratic (Q).b Mean for unstressed control given for comparison purposes only and not includ

ad small necrotic areas on leaf edges, but those that were notre-treated with ALA had moderate damage and lost considerableortions of their foliage (Table 1). These results correlate well withhe findings of Hotta et al. (1998) who found that pre-treatment ofice seedlings with ALA in the range of 0.1–1 ppm by root soakingesulted in higher seedling survival compared to non-treated plantsollowing a chilling stress at 5 ◦C for 5 days. Similar results were alsoeported by Wang et al. (2004) who stated that the protection ofLA treatment on melon seedlings against chilling injury was obvi-us. They found that control plants were completely dehydratednd dead after chilling stress at 8 ◦C for 2 h, whereas the plants soilrenched with 10 ppm ALA showed slight injury symptoms in a few

eaves.Among the application methods employed, seed soaking and

oliar spray seemed to provide better protection against chillingtress compared to soil drench. Even though seed soaking and foliarpray provided similar means of protection, soaking the seeds may

able 5ffect of ALA applications through different methods on proline, sucrose and potassiumubjected to chilling stress. Values are means ± SE (n = 8).

Treatments Proline (�mol g−1 FW) Sucr

App. methodSeed soak 0.51 ± 0.01 261Foliar spray 0.50 ± 0.02 247Soil drench 0.47 ± 0.01 254

ALA Con. (ppm)0 0.47 ± 0.01 202 ±1 0.48 ± 0.01 25510 0.49 ± 0.02 24825 0.56 ± 0.04 310 ±50 0.48 ± 0.01 255

Significancea L*Q** NSr2 0.60 –

Unstressed controlb 0.46 ± 0.02 299

ANOVAApp. method (M) NS NSALA Con. (C) ** **M × C NS NS

S, *, **, not significant, significant at P < 0.05 or 0.01, respectively.a Linear (L) or quadratic (Q).b Mean for unstressed control given for comparison purposes only and not included in

the statistical analysis.

be the simplest and most convenient method in providing chillingstress tolerance. This finding may especially be significant to thosewho has to grow peppers in large areas and do not wish to spraythe plants with ALA in the field.

The present study also revealed that the efficacy of ALA concen-trations in enhancing chilling tolerance leveled off at 25 ppm, sincethe relationship between ALA concentrations and majority of vari-ables measured was curvilinear. It is a well documented fact thatALA applied over certain concentrations is detrimental to plants,acting as herbicide (Chakraborty and Tripathy, 1992; Kumar et al.,1999). Thus, determination of an optimum concentration is a pre-requisite if ALA is to be used in enhancing plant growth under

optimum or stressful conditions.

Numerous studies have reported that exogenous applicationof ALA could promote growth and increase yield of several cropspecies. For example, Hotta et al. (1997) found that ALA appli-cation at low concentrations could significantly increase yield of

contents and superoxide dismutase (SOD) enzyme activity of pepper seedlings

ose (mg g−1 DW) SOD (U g−1 FW) K (ppm)

± 20 192 ± 12 448 ± 6± 17 200 ± 15 460 ± 7± 18 175 ± 11 434 ± 5

16 153 ± 15 433 ± 9± 19 178 ± 15 451 ± 6± 29 187 ± 13 452 ± 8

25 236 ± 21 450 ± 8± 24 191 ± 13 450 ± 8

L*Q** NS0.70 –

± 43 150 ± 20 447 ± 7

NS **** NSNS NS

the statistical analysis.

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arley, garlic, kidney bean, potato and radish, and they assumedhat higher productivity was related to increased photosynthe-is in light and decreased respiration in darkness. Memon et al.2009) reported that ALA treatment improved the biosynthesis ofhlorophyll which, in turn, increased photosynthetic capacity inakchoi. ALA is the key precursor in the biosynthesis of all por-hyrins compounds such as chlorophyll, heme, and phytochrome,nd its formation in plants is the rate-limiting step in tetrapyrroliosynthesis (von Wettstein et al., 1995; Wang et al., 2005). It is alsonown that ALA production in plants is sensitive to temperaturextremes, low and high temperatures inhibiting its biosynthesisHodgins and Öquist, 1989; Tewari and Tripathy, 1998). In thistudy, ALA pre-treatment enhanced Chl a and Chl b contents com-ared to non-ALA-treated plants under chilling stress (Table 2);hus, exogenous application of ALA prior to chilling stress could beway to overcome inadequate biosynthesis problem. Moreover, we

ound that ALA treatment prior to chilling stress elevated the pro-ine levels in pepper seedlings (Table 5) which might augment thehilling tolerance of pepper seedlings as an important osmoregula-or helping protect plant’s tissues from damaging effects of chillingtress (Ashraf and Foolad, 2007).

Exogenous application of ALA has several physiological and bio-hemical effects, including regulation of photosynthesis in plantsrown under normal or stressful conditions. Wang et al. (2004) doc-mented that the growth promotion in melon seedlings by ALAnder chilling stress might result from the increase of leaf pho-osynthesis. They found that ALA-treated plants had up to 200%reater leaf photosynthesis depending on the leaf position and7% greater plant dry weight compared to control plants. Theylso reported that stomatal conductance of ALA-treated plants wasbout two-fold as great as that of non-ALA-treated plants. Hottat al. (1997) documented that treatment of radish plants with ALAncreased photosynthesis by 12% and decreased dark respirationy 30%, both of which resulted in 30% yield increase. Promotionf stomatal conductance and photosynthesis is also reported inakchoi grown under normal conditions (Memon et al., 2009). Inur study, chlorophyll content (Table 2) and stomatal conductanceTable 3) increased considerably in response to ALA application.dditionally, although not statistically significant, increased K+

ontent of pepper seedlings in response to ALA application mayave affected stomatal conductivity, since K is known to regulatetomatal opening and closure (Nabors, 2004). The fact that non-LA-treaded plants had considerably lower K+ content compared

o ALA-treated plants and unstressed control plants shows thatLA pre-treatment improved K+ uptake under chilling stress con-itions. Higher chlorophyll content and stomatal conductance mayave caused higher shoot fresh and dry weights and sucrose con-ent since all these variables are closely related to photosyntheticapacity and dry matter production.

Treating plants with such plant growth substances as abscisiccid or salicylic acid could enhance chilling tolerance by inductionf gene expression (Kang et al., 2003; Warren, 2001). However,he enhanced chilling tolerance that benefit from the applicationf these compounds is usually offset by a reduction in stomatalonductance and transpiration which finally leads to reduced pho-osynthesis and growth (Janda et al., 1999; Liang et al., 1997). Onhe contrary, the application of ALA in this study did not reduce butather improved stomatal conductance and plant growth as wells chilling tolerance. Thus, as suggested by others too (Wang et al.,004), ALA could be used to improve chilling tolerance of plantsithout any adverse effect.

The most common visible symptom of low temperature stressn intact plant tissues is water loss, which finally leads to wilt-ng during and after the low temperature exposure. Wilting thatccurs during and/or after low temperature exposure is attributedo a primary mechanism involving loss of membrane properties or

rimental Botany 67 (2010) 495–501

transition of membranes from a normal fluid state to a restricted,less fluid, semi-crystalline state (Wright, 1974). Electrolyte leak-age reflects the extent of cell membrane injury. We observed thatplants pre-treated with ALA exhibited lower electrolyte leakagethereby higher RWC compared to those not pre-treated with ALA(Table 3) which confirms the findings of other researchers. Forexample, Hotta et al. (1998) reported that the pre-treatment of riceseedlings by root soaking with ALA solution reduced the ratio of leafrolling and electrolyte leakage from leaf tissues after a cold stresstreatment.

Antioxidant systems in plants prevent or mitigate the mem-brane peroxidation resulting from reactive oxygen species (ROS)under stressful conditions such as chilling, drought or salinity(Xu et al., 2008). SOD enzyme is located in the chloroplast, mito-chondrion, cytoplasm and peroxisome, and operates as the firstline of defense against ROS (Liau et al., 2007). Higher SOD activ-ity has been reported in many species under stress, and this wasattributed to de novo synthesis of enzymic protein (Song et al.,2006). ALA has been reported to stimulate the activities of antiox-idative enzymes including SOD in spinach seedlings subjected tosalinity stress (Nishihara et al., 2003) and pakchoi seedlings grownunder optimum conditions (Memon et al., 2009). Our findings alsoshow ALA-treated plants had enhanced SOD activity under chillingstress conditions (Table 5), which is well in line with the resultsmentioned above.

5. Conclusion

The result of the present study revealed that pre-treatment withALA through seed soaking, foliar spray, or soil drench was effectivein inducing chilling tolerance in pepper seedlings. Among the appli-cation methods employed, seed soaking and foliar spray providedbetter protection against chilling stress compared to soil drenching.Even though seed soaking and foliar spray provided similar meansof protection, seed soaking may be the simplest and most conve-nient method in providing chilling stress tolerance. There was acurvilinear relationship between ALA concentrations and chillingtolerance, and the best protection was obtained from plants pre-treated with 25 ppm ALA. Our finding that ALA could be used asa seed treatment to prevent crop losses in pepper due to chillingstress may have significant practical applications.

Acknowledgements

This work was supported by a grant (project no: 107O611) fromThe Scientific and Technical Research Council of Turkey (TUBITAK)and we are grateful for the financial support. We also thank Dr.M. Nuri Nas and Dr. Ferit Kocacınar for their critical review of themanuscript.

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