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Effects of Calcium, Stress onContents of Chlorophyll andCarotenoid, LPO Levels, andAntioxidant Enzyme Activitiesin MenthaNilgün Candan a & Leman Tarhan ba Department of Chemistry, Faculty of Art andScience , Dokuz Eylul University , Buca, İzmir,Turkeyb Department of Chemistry, Faculty of Education ,Dokuz Eylul University , Buca, İzmir, TurkeyPublished online: 14 Feb 2007.

To cite this article: Nilgün Candan & Leman Tarhan (2005) Effects of Calcium, Stresson Contents of Chlorophyll and Carotenoid, LPO Levels, and Antioxidant EnzymeActivities in Mentha , Journal of Plant Nutrition, 28:1, 127-139, DOI: 10.1081/PLN-200042192

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Journal of Plant Nutrition, 28: 127–139, 2005

Copyright © Taylor & Francis Inc.

ISSN: 0190-4167 print / 1532-4087 online

DOI: 10.1081/PLN-200042192

Effects of Calcium, Stress on Contentsof Chlorophyll and Carotenoid, LPO Levels,

and Antioxidant Enzyme Activities in Mentha

Nilgun Candan1 and Leman Tarhan2

1Department of Chemistry, Faculty of Art and Science and 2Department of Chemistry,Faculty of Education, Dokuz Eylul University, Buca, Izmir, Turkey

ABSTRACT

The distribution of chlorophyll and carotenoid contents and antioxidant enzyme activ-ities such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6),ascorbate-dependent peroxidase (AsA-dep POD, EC 1.11.1.11), guaiacol-dependentperoxidase (Gua-dep POD, EC 1.11.1.7), and lipid peroxidation levels (LPO) were in-vestigated according to the leaf positions of Mentha pulegium grown in the presence of4000 µM Ca2+ and in the absence of Ca2+ on the 14th day after the start of treatments.After reaching the highest values at leaf 6, chlorophyll-carotenoid content, SOD, CATactivities decreased as the leaves aged. The variations in chlorophyll-carotenoid contentsand antioxidant enzyme activities were investigated with decreasing Ca2+concentrationsranging from 2000 to 0 µM with respect to time. The chlorophyll-carotenoid values in-creased in the absence of Ca2+ approximately 4.5-fold compared to the control plantover the 19 days of the stress period. SOD activity also increased approximately 2-foldwith decreasing Ca2+ concentrations on the 14th day and decreased afterward. WhereasCAT, AsA-dep, and Gua-dep POD activities were observed under the control levelsup to 14th day and then increased over them. The LPO levels in the stress conditionsincreased with decreasing Ca2+ concentrations during the most of the treatment period.The peroxidation of polyunsaturated fatty acids in M. pulegium membranes was highlysuppressed by at least 100 µM Ca2+ concentration in the growth medium.

Keywords: antioxidant enzymes, Ca2+, chlorophyll, carotenoid, lipid peroxidation,Mentha pulegium

Received 22 July 2003; accepted 10 June 2004.Address correspondence to Leman Tarhan, Chemistry Department, Faculty of Edu-

cation, Dokuz Eylul University, Buca, 35150 Izmir, Turkey. E-mail: leman.tarhan@deu.edu.tr.

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INTRODUCTION

Plants are immobile and therefore have developed defense mechanisms to senseand respond to biotic and abiotic stresses so that they can better adapt to their en-vironment. Calcium appears to play a central role in many defense mechanismsinduced by stress, and Ca2+ signaling is required for acquisition of tolerance orresistance to the stress. Ca2+ is also thought to be involved in stimulating wallthickening, phytoallexin synthesis, and alteration of reactive oxygen species(ROS).

However, uncontrolled production of ROS (Halliwell, 1982), includingsuperoxide anion (O.−

2 ), hydroxyl radicals (OH.), singlet oxygen (.O2), and hy-drogen peroxide (H2O2), disturb the electron transport systems in chloroplastsand mitochondria. One of the most damaging effects of these molecular speciesand their products in cells is the peroxidation of membrane lipids. This pro-cess, as well as some of its by-products, may severely affect the functionaland structural integrity of biological membranes, resulting in an increase ofthe plasma membrane permeability, which leads to leakage of potassium ionsand other solutes (De Vos et al., 1989; Weckx and Clijsters, 1996) and mayfinally cause cell death (Tappel, 1973; Kappus, 1985). Plant cells are normallyprotected against harmful effects of active oxygen by a complex antioxidantsystem (Smirnoff, 1993; Candan and Tarhan, 2003); active oxygen species canbe scavenged by both enzymatic and nonenzymatic detoxification mechanisms(Breusegem et al., 1998). Superoxide dismutase (SOD), mostly localized inchloroplasts of leaves (Jackson et al., 1978), catalyses the dismutation of O.−

2 toH2O2 and O2, and the H2O2 is converted into O2 and H2O by a H2O2 scavengingenzyme, catalase (CAT) and various peroxidases (Lidon and Heriques, 1993;Pole, 1997). Susceptibility to oxidative stress depends on the overall balancebetween factors that increase oxidant generation and those cellular componentsthat exhibit on antioxidant capability (Halliwell and Gutteridge, 1989; Foyeret al., 1994).

Further insight into the nature of antioxidant defense systems will proveuseful in understanding both specific and general mechanisms of plant stress tol-erance, and thus it is important to investigate plant antioxidant defense-systemresponses to Ca2+ stress. In the present study, the responses of chlorophyll andcarotenoid content, antioxidant capacity, and LPO levels were investigated inleaf extracts of Mentha pulegium subjected to different concentrations of Ca2+

stress.

MATERIALS AND METHODS

Plant Growth Conditions

Seeds of Mentha pulegium L. were disinfected with 10% H2O2 for 20 min,washed thoroughly with distilled water, and germinated between wet paper

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towels at 25◦C in the dark for 3 days. Seedlings were grown in a growthchamber (16 h light/8 h dark) providing white fluorescent light (Philips, India)with 150 µmol.m−2.s−1 light intensity, day/night temperature of 25/20◦C, and65(±5)% relative humidity. The seedlings were grown in normal nutrient solu-tion until the number of leaves reached 8. The nutrient solution contained, perliter: KNO3 (1.02 g); Ca(NO3)2 (0.492 g); NH4H2PO4 (0.23 g); MgSO4.7H2O(0.49 g); H3BO3 (2.66 mg); MnCl2.4H2O (1.81 mg); CuSO4.5H2O (0.08 mg);ZnSO4.7H2O (0.22 mg); H2MoO4.H2O (0.09 mg); and 0.5% FeSO4.7H2O(0.6 mL) (Arnon and Hoagland, 1953). The solutions were continuouslyaerated and renewed 3 to 4 times a week to minimize pH shift and nutri-ent depletion. In the first step of the experiment, the seedlings were trans-ferred to a nutrient solution, which included 0 µM Ca2+ as absence con-ditions, 2000 µM Ca2+ as the control, and 4000 µM Ca2+ as excess con-ditions. After 14 days, treatments were performed; leaves were harvested,weighed, and used for the preparation of extracts for enzyme analysis. In asecond set of experiments, time-dependent variations were also investigated inleaves of M. pulegium grown in Ca2+ concentrations decreasing from 2000 to0 µM.

Leaf Position

Seedlings were selected for experiments based on their uniform appearance,in terms of the average height of the plants, the total number of leaves presenton each plant, and the size of leaves. The leaves at the shoot apex were con-sidered as leaf position 2 (leaf 2). Leaves 4, 6, and 8 were labeled for all theseedlings, counting down from the top of the plants at the beginning of the timetreatments.

Enzyme Determinations

Extracts of M. pulegium leaves were prepared for enzyme determinations. One gleaf material (without the main midribs) was homogenized in 4 mL 20 mM phos-phate buffer (pH 7.4) containing 50 mM β-mercaptoethanol. The homogenatewas filtered and then centrifuged at 15000 × g for 15 min. The supernatantwas used for enzyme analysis. All operations (until the enzyme determination)were done at 0 to 4◦C. The β-mercaptoethanol was not included in the homog-enizing buffer for determinations of Gua-dep POD activity and LPO levels.SOD assay was based on the inhibitory effects of SOD on the spontaneousautoxidation of 6-hydroxidopamine (Crosti et al., 1987). An autoxidation rateof 6-OHDA 0.4 mM in 0.1 M phosphate buffer (pH 7.4) that was saturatedby air-O2 (8.2 mg/l) was determined by observing the absorbance changes at490 nm in 15 sec time intervals at 25◦C. SOD activity assays were carried

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out by adding the amount of enzyme solution required to halve the initial ab-sorbance value of 6-OHDA autoxidation at the 90th sec. One IU is the amount ofSOD required to inhibit the initial rate of 6-hydroxydopamine autoxidation by50%.

Activity of AsA-dep POD was measured according to Nakano and Asada(1981) by monitoring the rate of ascorbate oxidation at 290 nm (E = 2.8 mM−1

cm−1). The reaction mixture contained 25 mM phosphate buffer (pH 7.0), 0.1mM EDTA, 1 mM H2O2, 0.25 mM AsA, and the enzyme sample at 25◦C. Nochange in absorption was found in the absence of AsA in the test medium. ForAsA-dep POD, 1 IU represents the amount of enzyme catalyzing the conversionof 1 µmol of substrate per minute.

For the measurement of Gua-dep POD activity, the reaction mixture con-tained 25 mM phosphate buffer (pH 7.0), 0.05% guaiacol, 10 mM H2O2, andthe enzyme sample. Activity was determined by the increase in absorbance at470 nm due to guaiacol oxidation at 25◦C (E = 26.6 mM−1 cm−1) (Nakanoand Asada, 1981). For PODs, 1 IU represents the amount of enzyme catalyzingthe conversion of 1 µmol of substrates per minute.

CAT activity was assayed in a reaction mixture containing 25 mM phos-phate buffer (pH 7.0) 10.5 mM H2O2, and enzyme. The decomposition of H2O2

was followed at 240 nm (E = 39.4 mM−1 cm−1) (Aebi, 1983). One IU of theenzyme activity was accepted as the amount of the enzyme, which decomposes1 µmol H2O2 per min at 25◦C.

Analytical Methods

Lipid peroxidation was estimated based on thiobarbituric acid (TBA) reactiv-ity. Samples were evaluated for malondialdehyde (MDA) production using aspectrophotometric assay for TBA. The extinction coefficient at 532 nm of153 mM−1 · cm−1 for the chromophore was used to calculate the MDA-likeTBA (Buege and Aust, 1978).

Concentrations of Chl (a + b) and carotenoid were measured as de-scribed by Lichtenthaler and Welburn (1983) after extraction with 80%acetone.

The total protein content was determined by the method of Bradford (1976)using bovine serum albumin (BSA) as standard (data not shown).

Statistical Analysis

A Tukey test was used for statistical significance analyses. The values are themean of three separate experiments. In addition, a comparison was made withPearson correlation for each substrate and/or enzyme.

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RESULTS

Variations in Chlorophyll and Carotenoid Contents, Antioxidant EnzymeActivities, and LPO Levels at Different Leaf Positions of M. pulegium

Under Ca2+ Stress

The antioxidant enzyme activities, such as SOD, CAT, AsA-dep POD, andGua-dep POD, and LPO levels were investigated in different leaf positions ofM. pulegium grown in the presence of 4000 µM Ca2+ as excess conditions,2000 µM Ca2+ as control, and 0 µM Ca2+ as absence conditions on the 14thday after the start of treatments (Fig. 1). Chlorophyll and carotenoid contentsin Ca2+-excess and -deficient treatments were increased when compared to thecontrol (P < 0.01), except for chlorophyll content in 4000 µM Ca2+ stressconditions.

Chlorophyll and carotenoid content and SOD and CAT activities reachedthe maximum values at leaf 6 for all Ca2+ stress conditions and decreased onleaf position 8 (p < 0.05) (Fig. 1, 2 A, B). The maximum SOD activities inleaf 6 in the absence of Ca2+ at 4000 µM and 2000 µM Ca2+ were determinedas 95.6 ± 2.8, 51.0 ± 1.7, and 47.0 ± 1.3 IU mg−1 protein, respectively. Themaximum CAT activities in leaf 6 in the absence of Ca2+ at 2000 µM and4000 µM Ca2+ were determined as 11.5 ± 0.2, 8.2 ± 0.1, and 6.74 ± 0.1 IUmg−1 protein, respectively.

AsA-dep POD activities showed no drastic change up to leaf 6 (p > 0.01,Fig. 2C) and reached the highest values in leaf 8, which are 4.74-fold at 4000 µMCa2+ and 9.57-fold in the absence of Ca2+ compared to that of the control(p < 0.01). Gua-dep POD activities tended to increase from the youngest tothe oldest leaves in Ca2+-stress conditions, but did not reach the level ofthe control (Fig. 2D) (p < 0.01). LPO levels in both Ca2+ stress conditions

Figure 1. Variations of total chlorophyll (A), carotenoid content (B) depending on theleaf positions of M. pulegium at the 14th day control, 2000 µM Ca2+ (-�-), 4000 µMCa2+ (- �-) and absence of Ca2+ (-�-).

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Figure 2. Variations of SOD (A), CAT (B), AsA-dep POD (C), Gua-dep POD (D) andLPO levels (E) depending on the leaf positions of M. pulegium at the 14th day control,2000 µM Ca2+ (-�-), 4000 µM Ca2+ (- �-) and absence of Ca2+ (-�-).

were higher than in the control and the highest LPO values were obtained at4000 µM Ca2+ (Fig. 2E, p < 0.01).

Based on high antioxidant enzyme activities in the above experiments, leaf6 of control group was selected for a second set of experiments on optimizationand time-related relationships.

Chlorophyll and Carotenoid Content in Ca2+ Deficiency Conditionswith Respect to Time

The Ca2+ deficiency resulted in severe suppression of leaf expansion and to-tal leaf area when compared with control (data not shown). Leaf numbers

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Calcium, Antioxidant Enzymes, Lipid Peroxidation 133

Figure 3. The variations of total chlorophyll (A) and carotenoid (B) in leaf 6 of M.pulegium in Ca2+ deficiency conditions depending on the time: ( -�-) 0 µM, (-•-)50 µM, (-◦-) 100 µM, (-�-) 1500 µM, and control medium (-�-) 2000 µM Ca2+.

were unchanged during the experimental period, whereas leaf thickness ofM. pulegium decreased slightly under Ca2+ deficiency (data not shown). Con-tents of chlorophyll and carotenoid changed similarly over the treatment period,while the contents in the absence of Ca2+ increased significantly from 11.46 ±0.32 to 51.38 ± 0.62 µg/cm2 and from 0.82 ± 0.01 to 4.06 ± 0.07 µg/cm2,respectively (p < 0.01) (Fig. 3).

Chlorophyll contents in Ca2+ concentrations ranging from 100 to 0 µMincreased significantly with respect to time (p < 0.001) and therefore the leavesappeared dark green, but some necrotic margins were observed in the middleand basal leaf blades of young leaves.

Activities of Radical Scavenging Enzymes and LPO Levels with Respectto Time Under Ca2+ Deficiency

Time-dependent variations in SOD activities were investigated in leaf 6 of M.pulegium grown in Ca2+ concentrations decreasing from 2000 µM to 0 µM.The maximum activities in Ca2+ concentrations ranging from 1500 to 0 µMincreased significantly from 48.01 ± 0.93 to 95.6 ± 2.81 IU mg−1 on the 14th

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Figure 4. SOD activity variations (A) in leaf 6 of M. pulegium in Ca2+ deficiencyconditions depending on time: (-�-) 0 µM, (-�-) 20 µM, (- �-) 50 µM, (-◦-) 100 µM,(-�-) 500 µM, (-�-) 1500 µM, and (-�-) 2000 µM Ca2+ (B) SOD activities dependingon Ca2+ concentrations on 14th day.

day and decreased significantly afterward (Fig. 4). The results of this studyshowed a negative correlation between SOD activity and Ca2+ concentrationthrough the treatment period (r = −0.516).

CAT activities in all Ca2+ conditions decreased up to the 14th day, afterwhich the activity change in the range of 0–100 µM Ca2+ displayed a sharpincrease, while the changes were less pronounced in the range of 500–1500µM Ca2+ when compared with the control (Fig. 5) (p < 0.01).

AsA-dep POD activities in the presence of 500 and 1500 µM Ca2+ con-centrations were similar to those of the control (p > 0.01), while lower activity

Figure 5. CAT activity variations (A) in leaf 6 of M. pulegium in Ca2+ deficiencyconditions depending on time: (-�-) 0 µM, (-�-) 20 µM, (- �-) 50 µM, (-◦-) 100 µM,(-�-) 500 µM, (-�-) 1500 µM, and (-�-) 2000 µM Ca2+ (B) CAT activities dependingon Ca2+ concentrations on 14th day.

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Calcium, Antioxidant Enzymes, Lipid Peroxidation 135

Figure 6. AsA-dep POD activity variations (A) in leaf 6 of M. pulegium in Ca2+ de-ficiency conditions depending on time: (-�-) 0 µM, (-�-) 20 µM, (-•-) 50 µM, (-◦-)100 µM, (-�-) 500 µM, (-�-) 1500 µM, and (-�-) 2000 µM Ca2+ (B) AsA-dep PODactivities depending on Ca2+ concentrations on 14th day.

values were observed in the range of 0–100 µM Ca2+ on the 14th day, afterwhich the activities increased significantly (Fig. 6).

A transient decrease in Gua-dep POD activities occurred during the 14 days,then the activities increased above the control level in the range of 0–100 µMCa+2 (p < 0.01) and approached the control level in other Ca+2 concentrationson the 19th day (p > 0.05) (Fig. 7).

LPO levels in Ca2+ deficiency conditions were higher than those of thecontrol, while a transient decrease in LPO levels was observed in the wholerange of Ca2+ concentrations for the first 7 days, then increased afterwards(Fig. 8).

Figure 7. Gua-dep POD activity variations (A) in leaf 6 of M. pulegium in Ca2+ de-ficiency conditions depending on time: (-�-) 0 µM, (-�-) 20 µM, (-•-) 50 µM, (-◦-)100 µM, (-�-) 500 µM, (-�-) 1500 µM, and (-�-) 2000 µM Ca2+ (B) Gua-dep PODactivities depending on Ca2+ concentrations on 14th day.

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Figure 8. Variations of LPO levels (A) in leaf 6 of M. pulegium in Ca2+ deficiencyconditions depending on time: (-�-) 0 µM, (-�-) 20 µM, (- �-) 50 µM, (-◦-) 100 µM,(-�-) 500 µM, (-�-) 1500 µM, and (-�-) 2000 µM Ca2+ (B) LPO levels depending onCa2+concentrations on 14th day.

DISCUSSION

Plants sense various biotic and abiotic stresses by Ca2+, which acts as an in-tracellular messenger in coupling a wide range of extracellular signals to spe-cific responses. The Ca2+ is implicated in regulating a number of fundamentalcellular processes that are involved in cytoplasmic streaming; cell division, dif-ferentiation, and polarity; and plant defense and stress responses (Reddy, 2001).Chlorophyll and carotenoid contents and antioxidant enzyme activities play animportant role in the cellular defense strategy against oxidative stress caused byvaried stress conditions. In the current study, the carotenoid contents and antiox-idant enzyme activities of SOD, CAT, and Gua-dep POD in all leaf positions ofM. pulegium grown in the absence of Ca2+ were found to be significantly higherthan the corresponding figures under excess Ca2+ conditions. These increasesprovided partial protection under absence conditions. Because lower LPO lev-els under these conditions were obtained when compared to excess conditions,it can also be argued that ROS generation and protection mechanism variationsagainst Ca-induced oxidative stress have important differences with respect toconcentrations of Ca2+ in the growth medium of M. pulegium. In plants, pow-erful homeostatic mechanisms must operate to keep cytosolic concentrationsof Ca2+ low; because it can be toxic, even small fluctuations in cytosolic Ca2+

concentrations drastically alter the activities of many enzymes.After reaching the highest values at leaf position 6, chlorophyll-carotenoid

content and SOD and CAT activities (r = −0.283, r = −0.277 and r = −0.479,r = −0.214) decreased as the leaves aged further. This situation is evidence ofincreased antioxidant defense systems against the Ca2+ stress conditions inthe development period of M. pulegium. Furthermore, the statistically higher

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LPO levels under excess Ca2+ that were observed in all leaf positions whencompared to the control may indicate that senescence processes are induced byCa2+ stress.

The antioxidant defense mechanism of the M. pulegium leaves (leaf 6)against Ca2+ deficiency showed different responses over time. During the first7 days, in spite of the decrease in CAT, AsA-dep, and Gua-dep POD activ-ities (r = −0.712, r = −0.586, r = −0.658; p < 0.001), LPO levels also de-clined (r = −0.658; p < 0.001) with increasing SOD activities and carotenoidcontents (r = 0.442, r = 0.804; p < 0.001). Carotenoids protect membranefrom photo-oxidation via scavenging effectively the singlet oxygen and ex-cited triplet chlorophyll. Similarly, SOD plays key roles in protecting the cellagainst the potentially deleterious effects of ROS. The increase of SOD ac-tivity suggests that levels of O.−

2 are increased in Mentha leaves in the Ca2+

deficiency. It is well documented that such protective antioxidant enzymes areactivated under stress conditions, which stimulate production of oxygen freeradicals. Increases in SOD activity were found in plants stressed by iron (Fe),copper (Cu), manganses (Mn), sulphur dioxide, and ozone toxicity (Tanakaand Sugahara, 1980; Hendry and Brocklebank, 1985; Tanaka et al., 1985;Candan and Tarhan, 2003a,b). However, the increase in SOD activities andcarotenoid contents during the first 7 days provided temporary defense againstCa2+ deficiency, because LPO levels began to increase significantly after the7th day even though SOD activities and carotenoid contents continued to in-crease. According to these findings, the increase in carotenoid content andSOD activities in M. pulegium leaves grown under Ca2+ deficiency condi-tions is not sufficient to protect the membrane against potentially deleteriouseffects of reactive oxygen species. The continuous and sharp decline of theH2O2 eliminating enzyme activities during these days may be the reason forthe increase in membrane damage caused by increasing levels of H2O2. Afterthe 14th day, the significant rise in H2O2 detoxifying enzymes with a nega-tive correlation with Ca2+ concentration can be explained by the role of theantioxidant defense mechanism of M. pulegium against Ca deficiency. How-ever, the decrease in the SOD activity seems to be a specific behavior inducedby senescence processes, since LPO levels increased sharply after the 14thday.

As a result of the response of M. pulegium antioxidant defense mech-anism against Ca deficiency in changing Ca2+ concentrations, CAT activityincreased, while SOD activity decreased up to 100 µM Ca2+ on the 14th dayand the maximum SOD activity value was observed in the absence of Ca2+.These results show that the increases of antioxidant enzyme activities are notsufficient for membrane protection, because membrane LPO level reached themaximum value in the absence of Ca2+. The increase in LPO level may indi-cate that at low Ca2+ concentrations, Fe+2 is released from exchange sites inliposomes, leading to a rise in .OH radical production via a metal-catalyzedHaber-Weiss reaction (Babizhayev, 1988). After decreasing sharply up to the

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100 µM Ca2+ concentration, LPO levels followed a similar trend in the rangeof 100–2000 µM. This indicates that inhibition of lipid peroxidation can beachieved by effective Ca2+ concentrations (100–2000 µM) through complexa-tion and thus suppression of the superoxide anion radicals.

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