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Antioxidative responses of Ocimum basilicum to sodium chloride or sodium sulphate salinization
Plant Physiology and Biochemistry 48 (2010) 772e777
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Research article
Antioxidative responses of Ocimum basilicum to sodium chloride or sodium sulphate salinization I. Tarchoune a, b, C. Sgherri b, *, R. Izzo b, M. Lachaal a, Z. Ouerghi a, F. Navari-Izzo b a b
Physiologie et Biochimie de la Tolérance au Sel des Plantes, Faculté des Sciences de Tunis, Campus Universitaire, 1060 Tunis, Tunisia Dipartimento di Biologia delle Piante Agrarie, Università di Pisa, Via del Borghetto 80, 56124 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 October 2009 Accepted 26 May 2010 Available online 9 June 2010
Soils and ground water in nature are dominated by chloride and sulphate salts. There have been several studies concerning NaCl salinity, however, little is known about the Na2SO4 one. The effects on antioxidative activities of chloride or sodium sulphate in terms of the same Naþ equivalents (25 mM Na2SO4 and 50 mM NaCl) were studied on 30 day-old plants of Ocimum basilicum L., variety Genovese subjected to 15 and 30 days of treatment. Growth, thiobarbituric acid reactive substances (TBARS), relative ion leakage ratio (RLR), hydrogen peroxide (H2O2), ascorbate and glutathione contents as well as the activities of ascorbate peroxidase (APX, EC 1.11.1.11); glutathione reductase (GR, EC 1.6.4.2) and peroxidases (POD, EC 1.11.1.7) were determined. In leaves, growth was more depressed by 25 mM Na2SO4 than 50 mM NaCl. The higher sensitivity of basil to Na2SO4 was associated with an enhanced accumulation of H2O2, an inhibition of APX, GR and POD activities (with the exception of POD under the 30-day-treatment) and a lower regeneration of reduced ascorbate (AsA) and reduced glutathione (GSH). However, the changes in the antioxidant metabolism were enough to limit oxidative damage, explaining the fact that RLR and TBARS levels were unchanged under both Na2SO4 and NaCl treatment. Moreover, for both salts the 30-day-treatment reduced H2O2 accumulation, unchanged RLR and TBARS levels, and enhanced the levels of antioxidants and antioxidative enzymes, thus achieving an adaptation mechanism against reactive oxygen species. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Antioxidative enzymes Ascorbate Basil Glutathione Hydrogen peroxide Sodium chloride Sodium sulphate
1. Introduction Salinity is one of the major environmental factors limiting global crop productivity, because it restricts crop yield particularly in the arid and semi-arid regions [30]. This is the case of Tunisia, an arid and semi-arid Mediterranean country, in which soils affected by salts cover about 1.5 million hectares, around 10% of the whole territory area. These saline soils are found throughout the country but especially in central and southern areas, where the
Abbreviations: APX, ascorbate peroxidase; AsA, ascorbate; DHA, dehydroascorbate; DTNB, 5,50 -dithiobis-nitrobenzoic acid; DW, dry weight; Na2EDTA, disodium ethylenediamine-tetraacetic acid; FW, fresh weight; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidised glutathione; H2O2, hydrogen peroxide; POX, peroxidase; RLR, relative ion leakage ratio; ROS, reactive oxygen species; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reacting substances; TCA, trichloroacetic acid. * Corresponding author. Tel.: þ39 50 2216633; fax: þ39 50 2216630. E-mail addresses: [email protected] (I. Tarchoune), [email protected]. it (C. Sgherri), [email protected] (R. Izzo), [email protected] (M. Lachaal), [email protected] (Z. Ouerghi). 0981-9428/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2010.05.006
inappropriate irrigation practices as well as the high evapo-transpiration rates are largely responsible for their extension. Salinity can affect growth and yield of plants and can also generate secondary oxidative stress that occurs when there is a serious imbalance between the production of reactive oxygen species (ROS) and antioxidant defense, leading to oxidative damage of macromolecules and, eventually, of cellular structure [17]. This is why ROS have been mainly considered as dangerous molecules, whose concentrations need to be maintained as low as possible. At different degrees, plant cells can tolerate ROS such as hydrogen peroxide (H2O2) and free radicals of O2 by enhancing endogenous protective mechanisms involving antioxidant molecules and enzymes. H2O2 is a strong nucleophilic oxidizing agent and the oxidation of SH-group is one major mode of its toxicity. In plant cells the ascorbate/glutathione cycle represents an alternative and more effective detoxification mechanism against H2O2 operating both in the chloroplasts and the cytosol [27]. It may remove H2O2 in a series of enzymatic reactions involving ascorbate peroxidase (APX) and glutathione reductase (GR). H2O2 is eliminated also by catalase and several classes of peroxidases (POD).
I. Tarchoune et al. / Plant Physiology and Biochemistry 48 (2010) 772e777
2. Results The concentrations used for NaCl and Na2SO4 represented the higher concentrations at which all plants were marketable, had a regular leaf shape and did not show any symptom of disorder. Growth of plants, estimated as FW and DW, was influenced in a different way by salts (Na2SO4 or NaCl, Table 1). In particular, after
15 days of treatment the 25 mM Na2SO4 reduced FW by 24%, whereas plants treated with 50 mM NaCl did not show significant differences in comparison with the control. Analogue behaviour was found for DW which showed a reduction by 45% in the sample treated with 25 mM Na2SO4 and no significant differences in the sample treated with 50 mM NaCl (Table 1). After 30 days of treatment FW decreased by 47% and 23% in plants treated with 25 mM Na2SO4 and 50 mM NaCl, respectively. DW values were halved by both treatments in comparison with the control. After 15 days of treatment leaf area (LA) decreased considerably by 35% in comparison with the control in plants treated with 25 mM Na2SO4 whereas remained unchanged in plants treated with 50 mM NaCl. After 30 days LA showed the maximum value in the control and decreased by 46% and 24% in plants treated with 25 mM Na2SO4 and 50 mM NaCl, respectively. Leaf number (Table 1) did not change either after 15 or 30 days, irrespective of treatment. RLR (Fig. 1A) and TBARS (Fig. 1B) did not show any change in plants treated both with Na2SO4 and NaCl, after 15 and 30 days of treatment. In contrast, H2O2 content (Fig. 1C) showed a significant increase both after 15 and 30 days of treatment in plants treated with Na2SO4 ( þ 108% and þ 46%, respectively). However, in plants treated with NaCl the increases were not significantly different from the control either after 15 or 30 days of treatment.
RLR (%100)
30
A
a
Control a a
Na2SO4 a
NaCl a
a
20
10
0 400 TBARS (nmol. g-1 DW)
The correlation between antioxidant capacity and salt tolerance is well known and has been demonstrated in numerous plant species. The overexpression of GR, POD and APX activities in Calendula officinalis leads to an increase in the resistance to oxidative stress [7] whereas the change in the levels of antioxidant molecules and enzyme activities in Helianthus annuus is among the first signals of plant tolerance and/or adaptation to stress conditions [9]. Generally, salt tolerance results from a greater efficiency of the antioxidative response under NaCl stress conditions [3] as the higher level of antioxidant enzyme activities in the salt-tolerant Lycopersicon pennellii is correlated to the inherently better protection from salt and oxidative stress [19]. In particular, the contents of the reduced ascorbate (AsA) and reduced glutathione (GSH) are crucial to ensure the defense against stress conditions [3,7,9]. Ocimum basilicum L. (Lamiaceae) is an important commercial plant, widely cultivated in many countries. It is also a medicinal plant used for several purposes, especially for therapeutical ones. Basil essential oil has been used extensively in the food industry as a flavouring agent, and in perfumery and medical industries [31]. It is also considered as a source of aroma compounds, and it possesses a range of biological activities being used as insect repellent, nematocidal, antibacterial, antifungal as well as antioxidant agent [15,31]. Despite the large body of literature on salt stress, few information exists for O. basilicum. Besides, soil salinity in nature is normally a mixture of different salts where sulphate and chloride salts often dominate [35]. Till now little attention has been given to salinities different from NaCl such as those caused by Na2SO4 [5,6]. The aim of this study was to investigate the different tolerance of O. basilicum L., variety Genovese, to two different salt stressors such as NaCl and Na2SO4. The study was performed in terms of equimolar concentrations of the two different sodium salts in order to evaluate the effects of the ions chloride and sulphate on growth and antioxidative response of basil. In particular, H2O2 production and antioxidative response were studied analyzing the changes in total and reduced ascorbate, in total and reduced glutathione as well as in the correlated antioxidative enzymes.
773
B
Control
300
Na2SO4 a
NaCl a
a
a
200
a
a 100
0
Treatments 25 mM Na2SO4
0 mM 15 days FW (g plant-1) DW (g plant-1) LA (cm2 plant-1) LN (number plant-1) 30 days FW (g plant-1) DW (g plant-1) LA (cm2 plant-1) LN (number plant-1)
50 mM NaCl
12.57 1.18 712 36
1.34 a 0.14 a 70.1 a 5.6 a
9.56 0.65 460 40
1.04 b 0.11b 8.40 b 3.1 a
11.68 1.03 632 40
0.22 a 0.03 a 78.4 a 5.8 a
33.74 4.44 1893 90
1.72 a 1.08 a 198.5 a 11.52 a
17.78 2.34 1014 80
0.71 c 0.32 b 106.4 c 10.55 a
25.97 2.84 1447 82
0.99 b 0.23 b 160.0 b 10.47 a
240 H2O2 (µmol. g-1 DW)
Table 1 Effect of salinity on growth parameters of basil (Ocimum basilicum L.), variety Genovese, treated with 25 mM Na2SO4 or 50 mM NaCl for 15 and 30 days. Data are the means of six independent experiments SE (n ¼ 6). Means followed by different letters are significantly different at P 0.05 as determined by Duncan’s multiple range test. FW, fresh weigh; DW, dry weight; LA, leaf area; LN, leaf number.
C
a
180
Control
Na2SO4
NaCl
ab a ab
120
b
b 60
0 15
30 Days of treatment
Fig. 1. Effect of 25 mM Na2SO4 or 50 mM NaCl on RLR (A), TBARS (B) and H2O2 content (C) in leaves of basil (Ocimum basilicum L.), variety Genovese, after 15 and 30 days of treatment. Means SE followed by different letters are significantly different at P 0.05 as determined by Duncan’s multiple range test.
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60 AsA + DHA (µmol. g-1 Dw)
After 15 days of treatment APX activity (Fig. 2A) decreased significantly by 58% in plants treated with 25 mM Na2SO4 compared to the control, whereas an increase by 55% was observed for plants treated with 50 mM NaCl. After 30 days APX activity increased both in plants treated with 25 mM Na2SO4 ( þ 60%) and with 50 mM NaCl ( þ 137%). After 15 days of treatment glutathione reductases GR activity (Fig. 2B) decreased in comparison with the control by 63% and 50% in plants treated with 25 mM Na2SO4 and 50 mM NaCl, respectively, whereas after 30 days GR activity remained constant with the 25 mM Na2SO4 treatment and increased 4 times with the 50 mM NaCl one. After 15 days of treatment POD activity (Fig. 2C) remained unchanged in comparison with the control when plants were treated with 50 mM NaCl. However, a decrease by 73% was monitored when plants were treated with 25 mM Na2SO4. After 30 days, POD activity increased by 255% in plants treated with 25 mM Na2SO4 and by 62% in those treated with 50 mM NaCl. Total ascorbate (AsA þ DHA) content (Fig. 3A) showed an increase by 28% after 15 days of treatment in plants treated with Na2SO4 and a decrease by 16% in plants treated with NaCl in comparison with the control. After 30 days of treatment increases by 39% and 59% were observed in the presence of 25 mM Na2SO4 and 50 mM NaCl, respectively. In comparison with the control, AsA content remained constant in plants treated for 15 days with
A
a Control b
40
APX (U.mg-1 protein)
Control
Na2SO4
NaCl
a
20
B
Control a
30
NaCl
a
a b
20
b
c
10
0.0 4.0
C
Control
Na2SO4
NaCl
a b
a b
2.0 b
b
a
b
6.0
b
0.0
4.0
15
Fig. 3. Effect of 25 mM Na2SO4 or 50 mM NaCl on total ascorbate (AsA þ DHA) (A), reduced (AsA) ascorbate (B) contents and reduced ascorbate (AsA)/dehydroascorbate (DHA) ratio (C) in leaves of basil (Ocimum basilicum L.), variety Genovese, after 15 and 30 days of treatment. Statistical analysis was as in Fig. 1.
B
Control
Na2SO4
NaCl a
0.3 a 0.2 b 0.1
c
b
b
0.0 25
C
30 Days of treatment
c
c
2.0
0.4 GR (U.mg-1 protein)
Na2SO4
1.0
0.0
POD (U.mg-1 protein)
b
c c
40
AsA / DHA
8.0
A
NaCl a
3.0 10
Na2SO4
0.0
AsA (µmol. g-1 Dw)
774
Control
a
a
Na2SO4
NaCl
20 a 15 10
b
b c
5.0 0.0 15
30 Days of treatment
Fig. 2. Effect of 25 mM Na2SO4 or 50 mM NaCl on the activities of APX (A), GR (B) and POD (C) in leaves of basil (Ocimum basilicum L.), variety Genovese, after 15 and 30 days of treatment. Statistical analysis was as in Fig. 1.
Na2SO4, whereas it decreased by 36% in those treated with NaCl. After 30 days of treatment increases by 15 and 36% were observed in the content of AsA in plants treated with 25 mM Na2SO4 and 50 mM NaCl, respectively. Notable is the decrease in the AsA/DHA ratio both after 15 and 30 days of treatment in the presence of 25 mM Na2SO4 (46% and 41%, respectively) and 50 mM NaCl (48% and 31%, respectively). The content of total glutathione (GSH þ GSSG) (Fig. 3A) showed a significant increase after 15 days of treatment both in the presence of 25 mM Na2SO4 ( þ 78%) and 50 mM NaCl ( þ 19%). After 30 days a reduction by 16% occurred in plants treated with 25 mM Na2SO4, whereas in those treated with 50 mM NaCl no significant difference was observed. GSH content (Fig. 3B), followed the same trend of total glutathione showing, after 15 days of treatment, increases by 95 and 27% for plants treated with 25 mM Na2SO4 and 50 mM NaCl, respectively. Moreover, after 30 days of treatment GSH level showed a decrease by 31% in the presence of 25 mM Na2SO4 and an increase by 19% in the presence of 50 mM NaCl in comparison with the control. GSH/GSSG ratio (Fig. 3C) remained unchanged after 15 days of treatment in comparison with the control for both salt treatments whereas, after 30 days, it underwent a significant decrease (30%) in plants treated with 25 mM Na2SO4 and an increase by 66% in those treated with 50 mM NaCl.
I. Tarchoune et al. / Plant Physiology and Biochemistry 48 (2010) 772e777
GSH+GSSG (µmol. g-1 Dw)
1.0
A
a
Control
Na2SO4
NaCl
0.8 b
c
0.5
a b
a
0.3
0.0 0.6 GSH (µmol. g-1 Dw)
The general effect of salinity is to reduce the growth rate due to osmotic and ionic effects [21]. Our results show that leaf plant biomass (FW and DW) and leaf area were particularly affected by the treatment with Na2SO4 whereas leaf number per plant remained unchanged with the salt treatment at both the two growth stages (Table 1). This finding suggests that salinity modified growth through its effects on the leaf expansion and not on the initiation of new leaves. Indeed, several authors have reported the strong implication of leaf expansion in glycophyte as well as in halophyte response to salinity [8]. Salt-induced growth reduction might be related to salt osmotic effects, which affect cell turgor and expansion [26]. Differently, Ben Amor et al. [2] reported that salinity affected initiation of new leaves without reducing cell expansion. Analysis of growth parameters (Table 1) is indicative of the fact that the inhibition effect of 25 mM Na2SO4 was stronger than that of 50 mM NaCl at both growth stages and that inhibition of growth was particularly shown by plants treated for 30 days. This, nothwithstanding the use of equimolar Naþ concentrations for Na2SO4 and NaCl. According to Munns [21] it may be explained with the fact that firstly salinity affects growth with a short-term effect, including osmotic effects, which reduce cell expansion. Second long-term effects, including excessive salt uptake, which cause plants’ suffering from ionic stress, take part afterwards. This leads to premature leaf aging which follows a reduction in the available photosynthetic area necessary to maintain growth. Based on cultivation with equimolar Naþ concentrations, similar findings were reported for potato [6] and for red-osier dogwood [25]. In contrast, in Sorghum bicolor [14], Quercus rubrus and Gossypium hirsutum [33], the inhibition effect of NaCl on growth was more pronounced than that of Na2SO4 . In Phaseolus vulgaris instead, both salts reduced growth rates to the same extent [16] suggesting that salt effects on plant growth might be species-specific. Salt stress has been widely shown to cause an increased generation of H2O2 responsible for lipid peroxidation in the absence of any protective mechanism [36]. In agreement with data on growth parameters, Na2SO4 induced an increased production of H2O2 at both growth stages compared to samples treated with NaCl (Fig. 1C) even if neither RLR nor TBARS, indicative of cellular damage, increased following the treatment (Fig. 1A, B). This suggests that in both cases the activation of an efficient free radical scavenging system could have minimized the adverse effects of a general peroxidation, thus contributing to the maintenance of membrane structure and integrity. Unchanged general peroxidation level and cell membrane stability seem to be characteristics of tolerant plants coping with salinity [7,23,29] being salt tolerance correlated with the stimulation of antioxidant enzymes and their enhanced ability to remove active oxygen species. Metabolism of hydrogen peroxide depends on various functionally interrelated antioxidant enzymes such as APX, GR and POD. In particular, APX play a key role in the removal of H2O2 in the chloroplast and cytosol [20,22], and changes in the activity of this enzyme are strictly correlated with plant tolerance to oxidative stress [3,7,23]. The increased production of H2O2 following a 15day-treatment with Na2SO4 could be due to a decreased APX activity (Figs. 1C and 2A) and this supports the hypothesis that basil was more affected by Na2SO4 stress compared to NaCl one. Indeed, NaCl did not significantly change the levels of H2O2 likely because of an induction of APX activity (Figs. 1C and 2A). Together APX, GR and POD activities were inhibited as well by a 15-day-treatment with Na2SO4 suggesting a general depression of the antioxidant enzymatic system in basil leaves at this stage. In contrast, the persisting oxidative conditions due to Na2SO4 treatment (30 days) induced APX and POD activities leading to
a reduced increase in H2O2 levels (Figs. 1C, 2A and 2C). Differences in antioxidant enzyme activation could be related to stress intensity [27] which depends on kind of salt and length of treatment (Fig. 2). Regeneration of oxidised glutathione by GR is a critical step of the ROS scavenging system. In this study, notwithstanding the decrease in foliar GR activity in Na2SO4 and NaCl-treated plants following a 15-day-treatment (Fig. 2B), an increase in GSH level (Fig. 4B) was observed suggesting that an enhanced GSH synthesis occurred. Indeed, increased amounts in total glutathione were detected as well (Fig. 4A). The very high relevant induction of GR activity found in the leaves of plants subjected to 50 mM NaCl and the higher GSH/GSSG ratio (Figs. 2B and 4C) found after 30 days can be correlated to acclimation or even tolerance to stress [10]. However, the unchanged GR value registered in the Na2SO4-treated plants (Fig. 2B) and the lower foliar GSH can be explained by an increased net glutathione degradation [22] or a decreased synthesis. AsA and GSH are the major redox buffer in the plant cell, present in almost all cell compartments and involved, in several cases, in the same physiological or defensive processes [22]. A 15-daytreatment with Na2SO4 enhanced foliar level of AsA þ DHA (Fig. 3A) and DHA without any significant variation in AsA (Fig. 3B) thus, a stimulation in AsA biosynthesis by Na2SO4 treatment can be suggested. In contrast, the decrease in AsA content under NaCl treatment could be explained with its consumption by APX for
B
Control a
0.4
Na2SO4
NaCl
a
b b b
c
0.2
0.0 3.0
GSH / GSSG
3. Discussion
775
C
Control
Na2SO4
NaCl a
2.0 a a
b a
c
1.0
0.0 15
30 Days of treatment
Fig. 4. Effect of 25 mM Na2SO4 or 50 mM NaCl on total glutathione (GSH þ GSSG) (A), reduced (GSH) glutathione (B) contents and reduced (GSH)/oxidized (GSSG) glutathione ratio (C) in leaves of basil (Ocimum basilicum L.), variety Genovese, after 15 and 30 days of treatment. Statistical analysis was as in Fig. 1.
776
I. Tarchoune et al. / Plant Physiology and Biochemistry 48 (2010) 772e777
detoxification of H2O2 (Figs. 2A, 3B and 1C). This suggests also that AsA levels were sufficient for maintaining APX activity, which can be irreversibly damaged when AsA concentration falls [1]. Moreover, the decrease in total ascorbate (Fig. 3A) suggests that utilization of AsA was higher than its synthesis. These results can explain the decline in AsA/DHA ratio (Fig. 3C) which is considered an important indicator of the cell redox status and one of the first sign of oxidative stress [28]. During persisting conditions of stress (30 days of treatment), the increase in total ascorbate and AsA (Fig. 3A, B) under both Na2SO4 and NaCl treatment and the increase AsA/DHA ratio (Fig. 3A) compared to that of the first period (15 days) suggest that plants adapt better to unfavourable conditions at this stage (30 days). GSH homeostasis in plants is essential for cellular redox control [29] and efficient responses to abiotic [17] and biotic stress [18]. In the leaves of basil, grown under 25 mM Na2SO4 and 50 mM NaCl for 15 days, the induction of glutathione biosynthesis (Fig. 4B) and the unchanged GSH/GSSG ratio (Fig. 4C), despite the increase in GSSG suggest that a sufficient counterbalance of GSH oxidation took place. The fact that after 30 days of treatment total glutathione remained unchanged in the NaCl-treated plants, whereas it decreased in the Na2SO4-treated ones (Fig. 4A), indicates once more that basil reacted better to NaCl than Na2SO4. In conclusion, the present study shows a different response in O. Basilicum to sodium chloride or sodium sulphate treatment, the latter leading to a stronger oxidative stress. Many defense strategies involving enzymatic and non-enzymatic components are functioning to ensure protection against reactive oxygen species. However, it is remarked that this antioxidative response was more efficient under NaCl than Na2SO4. 4. Materials and methods 4.1. Plant material and growth conditions Seeds of basil (O. basilicum L., variety Genovese) were germinated in the dark for 5 days in polyester shelf containing rockwool as inert substrate with distilled water in a growth chamber at 25/ 20 C (day/night). After one week, seedlings were irrigated with Hoagland’s solution [11]. After 15 days uniform seedlings were transferred on a floating polyester in plastic pots, at a density of 24 plants per pot, filled with 65 L of aerated Hoagland’s solution and renewed every two week. After 30 days, the plants were divided into three groups (two pots per treatment) and then exposed for 15 and 30 days to an equimolar concentration of different salt (25 mM Na2SO4 and 50 mM NaCl). Plant growth was carried out in MayeJuly 2007 in a greenhouse at a day/night temperature of 29/ 18 C, an average daily relative humidity of 70e90%, with a global daily radiation of 10 MJ/m2 and a photon flux density of 500e700 mmol m2 s1. 4.2. Growth parameters After 15 and 30 days of treatments (45- and 60-day-old-plants, respectively) six plants per treatment were harvested for growth measurements. Leaf fresh weight (FW) and dry weight (DW) (dried at 70 C for 48 h) of each plant were determined. Leaf area was measured using the Optimas software (Optimas corporation, OPTIMASÔ, version 6.1), after having scanned leaves of each plant. 4.3. Solute leakage Relative ion leakage ratio (RLR) was calculated as the ratio of conductivity (Metrohm 660 conductometer), after 1 h shaking of 25 leaf discs in 25 ml double-distilled water at room temperature, to
the conductivity of the same discs after treatment in liquid nitrogen and an additional hour shaking [24]. 4.4. Hydrogen peroxide H2O2 content was evaluated following the method reported by Sgherri and Navari-Izzo [27] based on the formation of the complex titanium-peroxide. Leaf tissue was homogenized with cold acetone and filtered. The precipitation of the peroxideetitanium complex was obtained by the addition to the extract of 5% titanyl sulphate and of concentrated NH4OH solution. After having washed 4 times the pellet with cold acetone, the precipitate was dissolved with 1.5 N H2SO4 and the solution read at 415 nm H2O2 content was calculated using a standard curve in the 0.5e10 mmol H2O2 range. According to the previous authors, this method is very sensitive and reproducible, and it excludes the interference of other peroxides except for a small amount of lipid peroxide. 4.5. Thiobarbituric acid reactive substances (TBARS) TBARS were determined according to Hodges et al. [12]. Fresh leaves were homogenized in 20% trichloroacetic acid (TCA) containing 0.5% thiobarbituric acid (1/12, w/v) and incubated at 95 C in a water bath for 30 min. Then, the mixture was quickly cooled in an ice-bath and centrifuged at 12 000 g for 15 min. To correct the measure for possible interference by malondialdehydeesugar complexes, which also absorb around 532 nm, an aliquot of the leaf extract was incubated without thiobarbituric acid (TBA) and the absorbance of the solution at 532 nm was subtracted from that containing TBA reagent. The absorbance of the sample was also read at 440 nm in addition to 532 and 600 nm. Calculations were performed using the following equation:
TBARS nmolm1 ¼ ððA BÞ=157; 000Þ 106 ; Where
A ¼ ½ðA532þTBA Þ ðA600þTBA Þ ðA532TBA A600TBA Þ and
B ¼ ½ðA440þTBA A600þTBA Þ0:0571
4.6. Ascorbate (AsA) and dehydroascorbate (DHA) Fresh leaf tissue was immediately homogenized at 4 C in 6% (w/v) trichloroacetic acid. After centrifugation at 12 100 g for 15 min, AsA and total AsA (AsA þ DHA) were determined in the supernatants according to the methods of Kampfenkel et al. [13] based on the reduction of Fe3þ by AsA, followed by complex formation between Fe2þ and 2,20 -dipyridyl, that absorbs at 525 nm. Total ascorbate was determined through a reduction of DHA to AsA by dithiothreitol. DHA content was estimated from the difference between total ascorbate and AsA. A standard curve covering the range of 5e50 nmol AsA was used. 4.7. Reduced glutathione (GSH) and oxidised glutathione (GSSG) Fresh leaf tissue was immediately homogenized at 4 C in 6% (w/v) TCA and centrifuged at 12 100 g for 15 min. The supernatant was used for total (GSH þ GSSG) and oxidised glutathione (GSSG) determination by the 5,50 - dithio-bis-nitro-benzoic acid (DTNB)GSSG reductase recycling procedure as reported in Sgherri and Navari-Izzo [27]. GSSG was determined after reduced glutathione
I. Tarchoune et al. / Plant Physiology and Biochemistry 48 (2010) 772e777
(GSH) had been removed by 2-vinylpyridine derivatizations. Changes in the absorbance of the reaction mixtures were measured at 412 nm at 25 C. The total glutathione contents were calculated from a standard calibration curve in which GSH equivalents (1e10 nmol) present were plotted against the rate of change in absorbance at 412 nm. 4.8. Enzyme extraction and activity Leaves from three separate plants for each treatment were immediately placed in liquid nitrogen and stored at 80 C prior to extraction. Leaf tissue (0.5 g) was ground in a cold mortar with sand and an appropriate cold extraction buffer at 0e4 C, as described below. The homogenates were centrifuged at 12 100 g for 15 min at 4 C. Enzyme assays were performed in the supernatant. The extraction for APX and POD was performed in 50 mM potassium phosphate (pH 7) containing 1 mM AsA. APX activity was measured by following the oxidation of AsA, operated by H2O2, at 290 nm and 25 C according to Wang and al [34]. POD activity was determined at 25 C following the increase in A430. One ml of reaction mixture contained 10 mM pyrogallol, 1.7 mM H2O2 and leaf extract in 0.1 M potassium phosphate (pH 6.5). Calculations were performed using an extinction coefficient of 2.47 mM1 cm1. The extraction of GR was performed in 0.1 M potassium phosphate (pH 7.5) containing 0.5 mM Na2EDTA, and its activity was assayed by the DTNB method, which was performed by following the increase in the absorbance at 412 nm and 30 C according to Smith and al. [32]. Proteins were determined according to Bensadoun and Weinstain [4] using serum bovine albumine as a standard. 4.9. Statistical analysis Results on growth parameters, RLR and TBARS are the means from two replicates of six independent experiments (n ¼ 6). Results on metabolites and enzymes are the means from two replicates of three independent experiments (n ¼ 3). All data are reported as mean value SE. One-way analysis of variance (ANOVA) was independently applied to the data to evaluate the salt-effect. Statistical assessments of differences between mean values were performed by Duncan’s multiple range test at P 0.05. When necessary, an arc sin or angular transformation was applied before statistical analysis. Acknowledgements This paper was supported by the University of Pisa (Fondi di Ateneo 2007e2008). Imen Tarchoune was supported by the Tunisian Ministry of Higher Education, Scientific Research and technology. References [1] K. Asada, The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 601e639. [2] N. Ben Amor, K. Ben Hamed, A. Debez, C. Grignon, C. Abdelly, Physiological and antioxidant responses of the perennial halophyte Crithmum maritimum to salinity. Plant Sci. 168 (2005) 889e899. [3] N. Ben Amor, A. Jiménez, W. Megdiche, M. Lundqvist, F. Sevilla, C. Abdelly, Response of antioxidant systems to NaCl stress in the halophyte Cakile maritime. Physiol. Plant 126 (2006) 446e457. [4] A. Bensadoun, D. Weinstein, Assay of proteins in the presence of interfering materials. Anal. Bioch. 70 (1976) 241e250. [5] Z. Bie, T. Ito, Y. Shinohara, Effects of sodium sulfate and sodium bicarbonate on the growth, gas exchange and mineral composition of lettuce. Sci. Hortic. 99 (2004) 215e224. [6] J.J. Bilski, D.C. Nelson, R.L. Conlon, The response of four potato cultivars to chloride salinity, sulfate salinity and calcium in pot experiments. Am. Potato J. 65 (1988) 85e90.
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[7] N. Chaparzadeh, M.L. D’Amico, R.A. Khavari-Nejad, R. Izzo, F. Navari-Izzo, Antioxidative responses of Calendula Officinalis under salinity conditions. Plant Physiol. Biochem. 42 (2004) 695e701. [8] P.S. Curtis, A. Läuchli, The role of leaf area development and photosynthetic capacity in determining growth of kenaf under moderate salt stress. Aust. J. Plant Physiol. 18 (1986) 553e565. [9] D. Di Baccio, F. Navari-Izzo, R. Izzo, Seawater irrigation: antioxidant defense responses in leaves and roots of a sunflower (Helianthus annuus L.) ecotype. J. Plant Physiol. 161 (2004) 1359e1366. [10] D.R. Gossett, E.P. Millhollon, M.C. Lucas, Antioxidant response to NaCl stress in salt-tolerant and salt-sensitive cultivars of cotton. Crop Sci. 34 (1994) 706e714. [11] D.R. Hoagland, D.I. Arnon, The water culture method for growing plants without soil. Cal. Agr. Exp. Stat. Cir. 347 (1950) 1e32. [12] D.M. Hodges, J.M. Delong, C.F. Forney, R.K. Prange, Improving the thiobarbituric acid reactive substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta. 207 (1999) 604e611. [13] K. Kampfenkel, M.V. Montagu, D. Inze, Extraction and determination of ascorbate and dehydroascorbate from plant tissue. Anal. Biochem. 225 (1995) 165e167. [14] A.H. Khan, M.Y. Ashraf, S.S.M. Naqvi, B. Khanzada, M. Ali, Growth, ion and solute contents of sorghum grown under NaCl and Na2SO4 salinity stress. Acta Physiol. Plant. 17 (1995) 261e268. [15] S.J. Lee, K. Umano, S.T. hibamoto, K.G. Lee, Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chem. 91 (2005) 131e137. [16] A. Meiri, J. Kamburoff, A. Poljakoff-Mayber, Response of bean plants to sodium chloride and sodium sulfate salinization. Ann. Bot. 35 (1971) 837e847. [17] S. Meneguzzo, F. Navari-Izzo, R. Izzo, Antioxidant responses of shoots and roots of wheat to increasing NaCl concentrations. J. Plant Physiol. 155 (1999) 274e280. [18] A. Meister, M.E. Anderson, Glutathione. Annu. Rev. Biochem. 52 (1983) 711e760. [19] V. Mittova, M. Volokita, M. Guy, M. Tal, Activities of SOD and the ascorbateglutathione cycle enzymes in subcellular compartments in leaves and roots of the cultivated tomato and its wild salt tolerant relative Lycopersicon pennellii. Physiol. Plant. 110 (2000) 42e51. [20] S. Morita, H. Kaminaka, T. Masumura, K. Tanaka, Induction of rice cytosolic ascorbate peroxidase mRNA by oxidative stress: the involvement of hydrogen peroxide in oxidative stress signalling. Plant Cell Physiol. 40 (1999) 417e422. [21] R. Munns, Comparative physiology of salt and water stress. Plant Cell Environ. 25 (2002) 239e250. [22] G. Noctor, C.H. Foyer, Ascorbate and glutathione: keeping active oxygen under control. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 249e279. [23] U. Pérez-López, A. Robredo, L.M. acuesta, C. Sgherri, A. Muñoz-Rueda, F. NavariIzzo, A. Mena-Petite, The oxidative stress caused by salinity in two barley cultivars is mitigated by elevated CO2. Physiol. Plant. 135 (2009) 29e42. [24] M.F. Quartacci, O. Glisi c, B. Stevanovi c, F. Navari-Izzo, Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration. J. Exp. Bot. 53 (2002) 2159e2166. [25] S. Renault, C. Croser, J.A. Franklin, J.J. Zwiazek, Effects of NaCl and Na2SO4 on red-osier dogwood (Cornus stolonifera Michx) seedlings. Plant. Soil. 233 (2001) 261e268. [26] J. Rozema, Growth, water and ion relationships of halophytic monocotyledonae and dicotyledonae: a unified concept. Aquat. Bot. 39 (1991) 3e16. [27] C.L.M. Sgherri, F. Navari-Izzo, Sunflower seedlings subjected to increasing water deficit stress: oxidative stress and defence mechanisms. Physiol. Plant. 93 (1995) 25e30. [28] C. Sgherri, M.F. Quartacci, R. Izzo, F. Navari-Izzo, Relation between lipoc acid and cell redox status in wheat grown in excess copper. Plant Physiol. Biochem. 40 (2002) 591e597. [29] A. Shalata, V. Mittova, M. Volokita, M. Guy, M. Tal, Response of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii to saltdependent oxidative stress: the root antioxidative system. Physiol. Plant. 112 (2001) 487e494. [30] M.C. Shannon, C.M. Grieve, L.E. Francois, Whole-plant response to salinity. in: R.E. Wilkinson (Ed.), PlanteEnvironment Interactions. Marcel Dekker, New York, 1994, pp. 199e224. [31] J.E. Simon, M.R. Morales, W.B. Phippen, R.F. Vieira, Z. Hao, A source of aroma compounds and a popular culinary and ornamental herb. in: J. Janick (Ed.), Perspectives on New Crops and New Uses. ASHS Press, Alexandria, VA, 1999, pp. 499e505. [32] I.K. Smith, T.L. Vierheller, C.A. Thorne, Assay of glutathione reductase in crude tissue homogenates using 5,5’-dithiobis (2-nitrobenzoic acid). Anal. Biochem. 175 (1988) 408e413. [33] B.P. Strogonov, Physiological basis of salt tolerance of plants (translated from the Russian) IPST, Jerusalem, 1962. [34] S.Y. Wang, H.J. Jiao, M. Faust, Changes in ascorbate, glutathione, and related enzymes activities during thidiazuron-induced bud break of apple. Physiol. Plant. 82 (1991) 231e236. [35] Z.Q. Wang., S.Q. Zhu, R.P. Yu, Salt-affected Soils in China, Sci.. press, Beijing, 1991. [36] L. Xiong, J.K. Zhu, Molecular and genetic aspects of plant responses to osmotic stress. Plant Cell Environ. 25 (2002) 131e139.
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Antioxidative responses of Ocimum basilicum to sodium chloride or sodium sulphate salinization I. Tarchoune a, b, C. Sgherri b, *, R. Izzo b, M. Lachaal a, Z. Ouerghi a, F. Navari-Izzo b a b
Physiologie et Biochimie de la Tolérance au Sel des Plantes, Faculté des Sciences de Tunis, Campus Universitaire, 1060 Tunis, Tunisia Dipartimento di Biologia delle Piante Agrarie, Università di Pisa, Via del Borghetto 80, 56124 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 October 2009 Accepted 26 May 2010 Available online 9 June 2010
Soils and ground water in nature are dominated by chloride and sulphate salts. There have been several studies concerning NaCl salinity, however, little is known about the Na2SO4 one. The effects on antioxidative activities of chloride or sodium sulphate in terms of the same Naþ equivalents (25 mM Na2SO4 and 50 mM NaCl) were studied on 30 day-old plants of Ocimum basilicum L., variety Genovese subjected to 15 and 30 days of treatment. Growth, thiobarbituric acid reactive substances (TBARS), relative ion leakage ratio (RLR), hydrogen peroxide (H2O2), ascorbate and glutathione contents as well as the activities of ascorbate peroxidase (APX, EC 1.11.1.11); glutathione reductase (GR, EC 1.6.4.2) and peroxidases (POD, EC 1.11.1.7) were determined. In leaves, growth was more depressed by 25 mM Na2SO4 than 50 mM NaCl. The higher sensitivity of basil to Na2SO4 was associated with an enhanced accumulation of H2O2, an inhibition of APX, GR and POD activities (with the exception of POD under the 30-day-treatment) and a lower regeneration of reduced ascorbate (AsA) and reduced glutathione (GSH). However, the changes in the antioxidant metabolism were enough to limit oxidative damage, explaining the fact that RLR and TBARS levels were unchanged under both Na2SO4 and NaCl treatment. Moreover, for both salts the 30-day-treatment reduced H2O2 accumulation, unchanged RLR and TBARS levels, and enhanced the levels of antioxidants and antioxidative enzymes, thus achieving an adaptation mechanism against reactive oxygen species. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Antioxidative enzymes Ascorbate Basil Glutathione Hydrogen peroxide Sodium chloride Sodium sulphate
1. Introduction Salinity is one of the major environmental factors limiting global crop productivity, because it restricts crop yield particularly in the arid and semi-arid regions [30]. This is the case of Tunisia, an arid and semi-arid Mediterranean country, in which soils affected by salts cover about 1.5 million hectares, around 10% of the whole territory area. These saline soils are found throughout the country but especially in central and southern areas, where the
Abbreviations: APX, ascorbate peroxidase; AsA, ascorbate; DHA, dehydroascorbate; DTNB, 5,50 -dithiobis-nitrobenzoic acid; DW, dry weight; Na2EDTA, disodium ethylenediamine-tetraacetic acid; FW, fresh weight; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidised glutathione; H2O2, hydrogen peroxide; POX, peroxidase; RLR, relative ion leakage ratio; ROS, reactive oxygen species; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reacting substances; TCA, trichloroacetic acid. * Corresponding author. Tel.: þ39 50 2216633; fax: þ39 50 2216630. E-mail addresses: [email protected] (I. Tarchoune), [email protected]. it (C. Sgherri), [email protected] (R. Izzo), [email protected] (M. Lachaal), [email protected] (Z. Ouerghi). 0981-9428/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2010.05.006
inappropriate irrigation practices as well as the high evapo-transpiration rates are largely responsible for their extension. Salinity can affect growth and yield of plants and can also generate secondary oxidative stress that occurs when there is a serious imbalance between the production of reactive oxygen species (ROS) and antioxidant defense, leading to oxidative damage of macromolecules and, eventually, of cellular structure [17]. This is why ROS have been mainly considered as dangerous molecules, whose concentrations need to be maintained as low as possible. At different degrees, plant cells can tolerate ROS such as hydrogen peroxide (H2O2) and free radicals of O2 by enhancing endogenous protective mechanisms involving antioxidant molecules and enzymes. H2O2 is a strong nucleophilic oxidizing agent and the oxidation of SH-group is one major mode of its toxicity. In plant cells the ascorbate/glutathione cycle represents an alternative and more effective detoxification mechanism against H2O2 operating both in the chloroplasts and the cytosol [27]. It may remove H2O2 in a series of enzymatic reactions involving ascorbate peroxidase (APX) and glutathione reductase (GR). H2O2 is eliminated also by catalase and several classes of peroxidases (POD).
I. Tarchoune et al. / Plant Physiology and Biochemistry 48 (2010) 772e777
2. Results The concentrations used for NaCl and Na2SO4 represented the higher concentrations at which all plants were marketable, had a regular leaf shape and did not show any symptom of disorder. Growth of plants, estimated as FW and DW, was influenced in a different way by salts (Na2SO4 or NaCl, Table 1). In particular, after
15 days of treatment the 25 mM Na2SO4 reduced FW by 24%, whereas plants treated with 50 mM NaCl did not show significant differences in comparison with the control. Analogue behaviour was found for DW which showed a reduction by 45% in the sample treated with 25 mM Na2SO4 and no significant differences in the sample treated with 50 mM NaCl (Table 1). After 30 days of treatment FW decreased by 47% and 23% in plants treated with 25 mM Na2SO4 and 50 mM NaCl, respectively. DW values were halved by both treatments in comparison with the control. After 15 days of treatment leaf area (LA) decreased considerably by 35% in comparison with the control in plants treated with 25 mM Na2SO4 whereas remained unchanged in plants treated with 50 mM NaCl. After 30 days LA showed the maximum value in the control and decreased by 46% and 24% in plants treated with 25 mM Na2SO4 and 50 mM NaCl, respectively. Leaf number (Table 1) did not change either after 15 or 30 days, irrespective of treatment. RLR (Fig. 1A) and TBARS (Fig. 1B) did not show any change in plants treated both with Na2SO4 and NaCl, after 15 and 30 days of treatment. In contrast, H2O2 content (Fig. 1C) showed a significant increase both after 15 and 30 days of treatment in plants treated with Na2SO4 ( þ 108% and þ 46%, respectively). However, in plants treated with NaCl the increases were not significantly different from the control either after 15 or 30 days of treatment.
RLR (%100)
30
A
a
Control a a
Na2SO4 a
NaCl a
a
20
10
0 400 TBARS (nmol. g-1 DW)
The correlation between antioxidant capacity and salt tolerance is well known and has been demonstrated in numerous plant species. The overexpression of GR, POD and APX activities in Calendula officinalis leads to an increase in the resistance to oxidative stress [7] whereas the change in the levels of antioxidant molecules and enzyme activities in Helianthus annuus is among the first signals of plant tolerance and/or adaptation to stress conditions [9]. Generally, salt tolerance results from a greater efficiency of the antioxidative response under NaCl stress conditions [3] as the higher level of antioxidant enzyme activities in the salt-tolerant Lycopersicon pennellii is correlated to the inherently better protection from salt and oxidative stress [19]. In particular, the contents of the reduced ascorbate (AsA) and reduced glutathione (GSH) are crucial to ensure the defense against stress conditions [3,7,9]. Ocimum basilicum L. (Lamiaceae) is an important commercial plant, widely cultivated in many countries. It is also a medicinal plant used for several purposes, especially for therapeutical ones. Basil essential oil has been used extensively in the food industry as a flavouring agent, and in perfumery and medical industries [31]. It is also considered as a source of aroma compounds, and it possesses a range of biological activities being used as insect repellent, nematocidal, antibacterial, antifungal as well as antioxidant agent [15,31]. Despite the large body of literature on salt stress, few information exists for O. basilicum. Besides, soil salinity in nature is normally a mixture of different salts where sulphate and chloride salts often dominate [35]. Till now little attention has been given to salinities different from NaCl such as those caused by Na2SO4 [5,6]. The aim of this study was to investigate the different tolerance of O. basilicum L., variety Genovese, to two different salt stressors such as NaCl and Na2SO4. The study was performed in terms of equimolar concentrations of the two different sodium salts in order to evaluate the effects of the ions chloride and sulphate on growth and antioxidative response of basil. In particular, H2O2 production and antioxidative response were studied analyzing the changes in total and reduced ascorbate, in total and reduced glutathione as well as in the correlated antioxidative enzymes.
773
B
Control
300
Na2SO4 a
NaCl a
a
a
200
a
a 100
0
Treatments 25 mM Na2SO4
0 mM 15 days FW (g plant-1) DW (g plant-1) LA (cm2 plant-1) LN (number plant-1) 30 days FW (g plant-1) DW (g plant-1) LA (cm2 plant-1) LN (number plant-1)
50 mM NaCl
12.57 1.18 712 36
1.34 a 0.14 a 70.1 a 5.6 a
9.56 0.65 460 40
1.04 b 0.11b 8.40 b 3.1 a
11.68 1.03 632 40
0.22 a 0.03 a 78.4 a 5.8 a
33.74 4.44 1893 90
1.72 a 1.08 a 198.5 a 11.52 a
17.78 2.34 1014 80
0.71 c 0.32 b 106.4 c 10.55 a
25.97 2.84 1447 82
0.99 b 0.23 b 160.0 b 10.47 a
240 H2O2 (µmol. g-1 DW)
Table 1 Effect of salinity on growth parameters of basil (Ocimum basilicum L.), variety Genovese, treated with 25 mM Na2SO4 or 50 mM NaCl for 15 and 30 days. Data are the means of six independent experiments SE (n ¼ 6). Means followed by different letters are significantly different at P 0.05 as determined by Duncan’s multiple range test. FW, fresh weigh; DW, dry weight; LA, leaf area; LN, leaf number.
C
a
180
Control
Na2SO4
NaCl
ab a ab
120
b
b 60
0 15
30 Days of treatment
Fig. 1. Effect of 25 mM Na2SO4 or 50 mM NaCl on RLR (A), TBARS (B) and H2O2 content (C) in leaves of basil (Ocimum basilicum L.), variety Genovese, after 15 and 30 days of treatment. Means SE followed by different letters are significantly different at P 0.05 as determined by Duncan’s multiple range test.
I. Tarchoune et al. / Plant Physiology and Biochemistry 48 (2010) 772e777
60 AsA + DHA (µmol. g-1 Dw)
After 15 days of treatment APX activity (Fig. 2A) decreased significantly by 58% in plants treated with 25 mM Na2SO4 compared to the control, whereas an increase by 55% was observed for plants treated with 50 mM NaCl. After 30 days APX activity increased both in plants treated with 25 mM Na2SO4 ( þ 60%) and with 50 mM NaCl ( þ 137%). After 15 days of treatment glutathione reductases GR activity (Fig. 2B) decreased in comparison with the control by 63% and 50% in plants treated with 25 mM Na2SO4 and 50 mM NaCl, respectively, whereas after 30 days GR activity remained constant with the 25 mM Na2SO4 treatment and increased 4 times with the 50 mM NaCl one. After 15 days of treatment POD activity (Fig. 2C) remained unchanged in comparison with the control when plants were treated with 50 mM NaCl. However, a decrease by 73% was monitored when plants were treated with 25 mM Na2SO4. After 30 days, POD activity increased by 255% in plants treated with 25 mM Na2SO4 and by 62% in those treated with 50 mM NaCl. Total ascorbate (AsA þ DHA) content (Fig. 3A) showed an increase by 28% after 15 days of treatment in plants treated with Na2SO4 and a decrease by 16% in plants treated with NaCl in comparison with the control. After 30 days of treatment increases by 39% and 59% were observed in the presence of 25 mM Na2SO4 and 50 mM NaCl, respectively. In comparison with the control, AsA content remained constant in plants treated for 15 days with
A
a Control b
40
APX (U.mg-1 protein)
Control
Na2SO4
NaCl
a
20
B
Control a
30
NaCl
a
a b
20
b
c
10
0.0 4.0
C
Control
Na2SO4
NaCl
a b
a b
2.0 b
b
a
b
6.0
b
0.0
4.0
15
Fig. 3. Effect of 25 mM Na2SO4 or 50 mM NaCl on total ascorbate (AsA þ DHA) (A), reduced (AsA) ascorbate (B) contents and reduced ascorbate (AsA)/dehydroascorbate (DHA) ratio (C) in leaves of basil (Ocimum basilicum L.), variety Genovese, after 15 and 30 days of treatment. Statistical analysis was as in Fig. 1.
B
Control
Na2SO4
NaCl a
0.3 a 0.2 b 0.1
c
b
b
0.0 25
C
30 Days of treatment
c
c
2.0
0.4 GR (U.mg-1 protein)
Na2SO4
1.0
0.0
POD (U.mg-1 protein)
b
c c
40
AsA / DHA
8.0
A
NaCl a
3.0 10
Na2SO4
0.0
AsA (µmol. g-1 Dw)
774
Control
a
a
Na2SO4
NaCl
20 a 15 10
b
b c
5.0 0.0 15
30 Days of treatment
Fig. 2. Effect of 25 mM Na2SO4 or 50 mM NaCl on the activities of APX (A), GR (B) and POD (C) in leaves of basil (Ocimum basilicum L.), variety Genovese, after 15 and 30 days of treatment. Statistical analysis was as in Fig. 1.
Na2SO4, whereas it decreased by 36% in those treated with NaCl. After 30 days of treatment increases by 15 and 36% were observed in the content of AsA in plants treated with 25 mM Na2SO4 and 50 mM NaCl, respectively. Notable is the decrease in the AsA/DHA ratio both after 15 and 30 days of treatment in the presence of 25 mM Na2SO4 (46% and 41%, respectively) and 50 mM NaCl (48% and 31%, respectively). The content of total glutathione (GSH þ GSSG) (Fig. 3A) showed a significant increase after 15 days of treatment both in the presence of 25 mM Na2SO4 ( þ 78%) and 50 mM NaCl ( þ 19%). After 30 days a reduction by 16% occurred in plants treated with 25 mM Na2SO4, whereas in those treated with 50 mM NaCl no significant difference was observed. GSH content (Fig. 3B), followed the same trend of total glutathione showing, after 15 days of treatment, increases by 95 and 27% for plants treated with 25 mM Na2SO4 and 50 mM NaCl, respectively. Moreover, after 30 days of treatment GSH level showed a decrease by 31% in the presence of 25 mM Na2SO4 and an increase by 19% in the presence of 50 mM NaCl in comparison with the control. GSH/GSSG ratio (Fig. 3C) remained unchanged after 15 days of treatment in comparison with the control for both salt treatments whereas, after 30 days, it underwent a significant decrease (30%) in plants treated with 25 mM Na2SO4 and an increase by 66% in those treated with 50 mM NaCl.
I. Tarchoune et al. / Plant Physiology and Biochemistry 48 (2010) 772e777
GSH+GSSG (µmol. g-1 Dw)
1.0
A
a
Control
Na2SO4
NaCl
0.8 b
c
0.5
a b
a
0.3
0.0 0.6 GSH (µmol. g-1 Dw)
The general effect of salinity is to reduce the growth rate due to osmotic and ionic effects [21]. Our results show that leaf plant biomass (FW and DW) and leaf area were particularly affected by the treatment with Na2SO4 whereas leaf number per plant remained unchanged with the salt treatment at both the two growth stages (Table 1). This finding suggests that salinity modified growth through its effects on the leaf expansion and not on the initiation of new leaves. Indeed, several authors have reported the strong implication of leaf expansion in glycophyte as well as in halophyte response to salinity [8]. Salt-induced growth reduction might be related to salt osmotic effects, which affect cell turgor and expansion [26]. Differently, Ben Amor et al. [2] reported that salinity affected initiation of new leaves without reducing cell expansion. Analysis of growth parameters (Table 1) is indicative of the fact that the inhibition effect of 25 mM Na2SO4 was stronger than that of 50 mM NaCl at both growth stages and that inhibition of growth was particularly shown by plants treated for 30 days. This, nothwithstanding the use of equimolar Naþ concentrations for Na2SO4 and NaCl. According to Munns [21] it may be explained with the fact that firstly salinity affects growth with a short-term effect, including osmotic effects, which reduce cell expansion. Second long-term effects, including excessive salt uptake, which cause plants’ suffering from ionic stress, take part afterwards. This leads to premature leaf aging which follows a reduction in the available photosynthetic area necessary to maintain growth. Based on cultivation with equimolar Naþ concentrations, similar findings were reported for potato [6] and for red-osier dogwood [25]. In contrast, in Sorghum bicolor [14], Quercus rubrus and Gossypium hirsutum [33], the inhibition effect of NaCl on growth was more pronounced than that of Na2SO4 . In Phaseolus vulgaris instead, both salts reduced growth rates to the same extent [16] suggesting that salt effects on plant growth might be species-specific. Salt stress has been widely shown to cause an increased generation of H2O2 responsible for lipid peroxidation in the absence of any protective mechanism [36]. In agreement with data on growth parameters, Na2SO4 induced an increased production of H2O2 at both growth stages compared to samples treated with NaCl (Fig. 1C) even if neither RLR nor TBARS, indicative of cellular damage, increased following the treatment (Fig. 1A, B). This suggests that in both cases the activation of an efficient free radical scavenging system could have minimized the adverse effects of a general peroxidation, thus contributing to the maintenance of membrane structure and integrity. Unchanged general peroxidation level and cell membrane stability seem to be characteristics of tolerant plants coping with salinity [7,23,29] being salt tolerance correlated with the stimulation of antioxidant enzymes and their enhanced ability to remove active oxygen species. Metabolism of hydrogen peroxide depends on various functionally interrelated antioxidant enzymes such as APX, GR and POD. In particular, APX play a key role in the removal of H2O2 in the chloroplast and cytosol [20,22], and changes in the activity of this enzyme are strictly correlated with plant tolerance to oxidative stress [3,7,23]. The increased production of H2O2 following a 15day-treatment with Na2SO4 could be due to a decreased APX activity (Figs. 1C and 2A) and this supports the hypothesis that basil was more affected by Na2SO4 stress compared to NaCl one. Indeed, NaCl did not significantly change the levels of H2O2 likely because of an induction of APX activity (Figs. 1C and 2A). Together APX, GR and POD activities were inhibited as well by a 15-day-treatment with Na2SO4 suggesting a general depression of the antioxidant enzymatic system in basil leaves at this stage. In contrast, the persisting oxidative conditions due to Na2SO4 treatment (30 days) induced APX and POD activities leading to
a reduced increase in H2O2 levels (Figs. 1C, 2A and 2C). Differences in antioxidant enzyme activation could be related to stress intensity [27] which depends on kind of salt and length of treatment (Fig. 2). Regeneration of oxidised glutathione by GR is a critical step of the ROS scavenging system. In this study, notwithstanding the decrease in foliar GR activity in Na2SO4 and NaCl-treated plants following a 15-day-treatment (Fig. 2B), an increase in GSH level (Fig. 4B) was observed suggesting that an enhanced GSH synthesis occurred. Indeed, increased amounts in total glutathione were detected as well (Fig. 4A). The very high relevant induction of GR activity found in the leaves of plants subjected to 50 mM NaCl and the higher GSH/GSSG ratio (Figs. 2B and 4C) found after 30 days can be correlated to acclimation or even tolerance to stress [10]. However, the unchanged GR value registered in the Na2SO4-treated plants (Fig. 2B) and the lower foliar GSH can be explained by an increased net glutathione degradation [22] or a decreased synthesis. AsA and GSH are the major redox buffer in the plant cell, present in almost all cell compartments and involved, in several cases, in the same physiological or defensive processes [22]. A 15-daytreatment with Na2SO4 enhanced foliar level of AsA þ DHA (Fig. 3A) and DHA without any significant variation in AsA (Fig. 3B) thus, a stimulation in AsA biosynthesis by Na2SO4 treatment can be suggested. In contrast, the decrease in AsA content under NaCl treatment could be explained with its consumption by APX for
B
Control a
0.4
Na2SO4
NaCl
a
b b b
c
0.2
0.0 3.0
GSH / GSSG
3. Discussion
775
C
Control
Na2SO4
NaCl a
2.0 a a
b a
c
1.0
0.0 15
30 Days of treatment
Fig. 4. Effect of 25 mM Na2SO4 or 50 mM NaCl on total glutathione (GSH þ GSSG) (A), reduced (GSH) glutathione (B) contents and reduced (GSH)/oxidized (GSSG) glutathione ratio (C) in leaves of basil (Ocimum basilicum L.), variety Genovese, after 15 and 30 days of treatment. Statistical analysis was as in Fig. 1.
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detoxification of H2O2 (Figs. 2A, 3B and 1C). This suggests also that AsA levels were sufficient for maintaining APX activity, which can be irreversibly damaged when AsA concentration falls [1]. Moreover, the decrease in total ascorbate (Fig. 3A) suggests that utilization of AsA was higher than its synthesis. These results can explain the decline in AsA/DHA ratio (Fig. 3C) which is considered an important indicator of the cell redox status and one of the first sign of oxidative stress [28]. During persisting conditions of stress (30 days of treatment), the increase in total ascorbate and AsA (Fig. 3A, B) under both Na2SO4 and NaCl treatment and the increase AsA/DHA ratio (Fig. 3A) compared to that of the first period (15 days) suggest that plants adapt better to unfavourable conditions at this stage (30 days). GSH homeostasis in plants is essential for cellular redox control [29] and efficient responses to abiotic [17] and biotic stress [18]. In the leaves of basil, grown under 25 mM Na2SO4 and 50 mM NaCl for 15 days, the induction of glutathione biosynthesis (Fig. 4B) and the unchanged GSH/GSSG ratio (Fig. 4C), despite the increase in GSSG suggest that a sufficient counterbalance of GSH oxidation took place. The fact that after 30 days of treatment total glutathione remained unchanged in the NaCl-treated plants, whereas it decreased in the Na2SO4-treated ones (Fig. 4A), indicates once more that basil reacted better to NaCl than Na2SO4. In conclusion, the present study shows a different response in O. Basilicum to sodium chloride or sodium sulphate treatment, the latter leading to a stronger oxidative stress. Many defense strategies involving enzymatic and non-enzymatic components are functioning to ensure protection against reactive oxygen species. However, it is remarked that this antioxidative response was more efficient under NaCl than Na2SO4. 4. Materials and methods 4.1. Plant material and growth conditions Seeds of basil (O. basilicum L., variety Genovese) were germinated in the dark for 5 days in polyester shelf containing rockwool as inert substrate with distilled water in a growth chamber at 25/ 20 C (day/night). After one week, seedlings were irrigated with Hoagland’s solution [11]. After 15 days uniform seedlings were transferred on a floating polyester in plastic pots, at a density of 24 plants per pot, filled with 65 L of aerated Hoagland’s solution and renewed every two week. After 30 days, the plants were divided into three groups (two pots per treatment) and then exposed for 15 and 30 days to an equimolar concentration of different salt (25 mM Na2SO4 and 50 mM NaCl). Plant growth was carried out in MayeJuly 2007 in a greenhouse at a day/night temperature of 29/ 18 C, an average daily relative humidity of 70e90%, with a global daily radiation of 10 MJ/m2 and a photon flux density of 500e700 mmol m2 s1. 4.2. Growth parameters After 15 and 30 days of treatments (45- and 60-day-old-plants, respectively) six plants per treatment were harvested for growth measurements. Leaf fresh weight (FW) and dry weight (DW) (dried at 70 C for 48 h) of each plant were determined. Leaf area was measured using the Optimas software (Optimas corporation, OPTIMASÔ, version 6.1), after having scanned leaves of each plant. 4.3. Solute leakage Relative ion leakage ratio (RLR) was calculated as the ratio of conductivity (Metrohm 660 conductometer), after 1 h shaking of 25 leaf discs in 25 ml double-distilled water at room temperature, to
the conductivity of the same discs after treatment in liquid nitrogen and an additional hour shaking [24]. 4.4. Hydrogen peroxide H2O2 content was evaluated following the method reported by Sgherri and Navari-Izzo [27] based on the formation of the complex titanium-peroxide. Leaf tissue was homogenized with cold acetone and filtered. The precipitation of the peroxideetitanium complex was obtained by the addition to the extract of 5% titanyl sulphate and of concentrated NH4OH solution. After having washed 4 times the pellet with cold acetone, the precipitate was dissolved with 1.5 N H2SO4 and the solution read at 415 nm H2O2 content was calculated using a standard curve in the 0.5e10 mmol H2O2 range. According to the previous authors, this method is very sensitive and reproducible, and it excludes the interference of other peroxides except for a small amount of lipid peroxide. 4.5. Thiobarbituric acid reactive substances (TBARS) TBARS were determined according to Hodges et al. [12]. Fresh leaves were homogenized in 20% trichloroacetic acid (TCA) containing 0.5% thiobarbituric acid (1/12, w/v) and incubated at 95 C in a water bath for 30 min. Then, the mixture was quickly cooled in an ice-bath and centrifuged at 12 000 g for 15 min. To correct the measure for possible interference by malondialdehydeesugar complexes, which also absorb around 532 nm, an aliquot of the leaf extract was incubated without thiobarbituric acid (TBA) and the absorbance of the solution at 532 nm was subtracted from that containing TBA reagent. The absorbance of the sample was also read at 440 nm in addition to 532 and 600 nm. Calculations were performed using the following equation:
TBARS nmolm1 ¼ ððA BÞ=157; 000Þ 106 ; Where
A ¼ ½ðA532þTBA Þ ðA600þTBA Þ ðA532TBA A600TBA Þ and
B ¼ ½ðA440þTBA A600þTBA Þ0:0571
4.6. Ascorbate (AsA) and dehydroascorbate (DHA) Fresh leaf tissue was immediately homogenized at 4 C in 6% (w/v) trichloroacetic acid. After centrifugation at 12 100 g for 15 min, AsA and total AsA (AsA þ DHA) were determined in the supernatants according to the methods of Kampfenkel et al. [13] based on the reduction of Fe3þ by AsA, followed by complex formation between Fe2þ and 2,20 -dipyridyl, that absorbs at 525 nm. Total ascorbate was determined through a reduction of DHA to AsA by dithiothreitol. DHA content was estimated from the difference between total ascorbate and AsA. A standard curve covering the range of 5e50 nmol AsA was used. 4.7. Reduced glutathione (GSH) and oxidised glutathione (GSSG) Fresh leaf tissue was immediately homogenized at 4 C in 6% (w/v) TCA and centrifuged at 12 100 g for 15 min. The supernatant was used for total (GSH þ GSSG) and oxidised glutathione (GSSG) determination by the 5,50 - dithio-bis-nitro-benzoic acid (DTNB)GSSG reductase recycling procedure as reported in Sgherri and Navari-Izzo [27]. GSSG was determined after reduced glutathione
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(GSH) had been removed by 2-vinylpyridine derivatizations. Changes in the absorbance of the reaction mixtures were measured at 412 nm at 25 C. The total glutathione contents were calculated from a standard calibration curve in which GSH equivalents (1e10 nmol) present were plotted against the rate of change in absorbance at 412 nm. 4.8. Enzyme extraction and activity Leaves from three separate plants for each treatment were immediately placed in liquid nitrogen and stored at 80 C prior to extraction. Leaf tissue (0.5 g) was ground in a cold mortar with sand and an appropriate cold extraction buffer at 0e4 C, as described below. The homogenates were centrifuged at 12 100 g for 15 min at 4 C. Enzyme assays were performed in the supernatant. The extraction for APX and POD was performed in 50 mM potassium phosphate (pH 7) containing 1 mM AsA. APX activity was measured by following the oxidation of AsA, operated by H2O2, at 290 nm and 25 C according to Wang and al [34]. POD activity was determined at 25 C following the increase in A430. One ml of reaction mixture contained 10 mM pyrogallol, 1.7 mM H2O2 and leaf extract in 0.1 M potassium phosphate (pH 6.5). Calculations were performed using an extinction coefficient of 2.47 mM1 cm1. The extraction of GR was performed in 0.1 M potassium phosphate (pH 7.5) containing 0.5 mM Na2EDTA, and its activity was assayed by the DTNB method, which was performed by following the increase in the absorbance at 412 nm and 30 C according to Smith and al. [32]. Proteins were determined according to Bensadoun and Weinstain [4] using serum bovine albumine as a standard. 4.9. Statistical analysis Results on growth parameters, RLR and TBARS are the means from two replicates of six independent experiments (n ¼ 6). Results on metabolites and enzymes are the means from two replicates of three independent experiments (n ¼ 3). All data are reported as mean value SE. One-way analysis of variance (ANOVA) was independently applied to the data to evaluate the salt-effect. Statistical assessments of differences between mean values were performed by Duncan’s multiple range test at P 0.05. When necessary, an arc sin or angular transformation was applied before statistical analysis. Acknowledgements This paper was supported by the University of Pisa (Fondi di Ateneo 2007e2008). Imen Tarchoune was supported by the Tunisian Ministry of Higher Education, Scientific Research and technology. References [1] K. Asada, The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 601e639. [2] N. Ben Amor, K. Ben Hamed, A. Debez, C. Grignon, C. Abdelly, Physiological and antioxidant responses of the perennial halophyte Crithmum maritimum to salinity. Plant Sci. 168 (2005) 889e899. [3] N. Ben Amor, A. Jiménez, W. Megdiche, M. Lundqvist, F. Sevilla, C. Abdelly, Response of antioxidant systems to NaCl stress in the halophyte Cakile maritime. Physiol. Plant 126 (2006) 446e457. [4] A. Bensadoun, D. Weinstein, Assay of proteins in the presence of interfering materials. Anal. Bioch. 70 (1976) 241e250. [5] Z. Bie, T. Ito, Y. Shinohara, Effects of sodium sulfate and sodium bicarbonate on the growth, gas exchange and mineral composition of lettuce. Sci. Hortic. 99 (2004) 215e224. [6] J.J. Bilski, D.C. Nelson, R.L. Conlon, The response of four potato cultivars to chloride salinity, sulfate salinity and calcium in pot experiments. Am. Potato J. 65 (1988) 85e90.
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