Lipoic acid mitigates oxidative stress and recovers metabolic distortions in salt-stressed wheat seedlings by modulating ion homeostasis, osmoregulator
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level and antioxidant system Zeynep GORCEK and Serkan ERDAL* Department of Biology, Science Faculty, Ataturk University, Erzurum, Turkey
* Correspondence Serkan ERDAL Tel: +90 442 231 4206 Fax: +90 442 236 0948 E-mail address: [email protected]
Abstract BACKROUND: Soil salinity is one of the most detrimental environmental factors affecting the growth and limiting the agricultural productivity of plants. This study investigated whether exogenous lipoic acid (LA) pretreatment plays a role in promoting salt tolerance in wheat seedlings. The seedlings were treated with LA (1.75 mM) and salt (100 mM NaCl) separately and their combination. RESULTS: Salt stress significantly reduced relative water content (RWC), leaf surface area, ribulosebisphosphate carboxylase (RuBisCo) expression, and chlorophyll content but increased the content of osmoregulators protein, carbohydrates, and proline. In addition, salinity led to an imbalance in the inorganic composition of wheat leaves.
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While it elevated Na+ content compared to control, Ca content and K+/Na+ ratio were reduced. Under saline conditions, despite increases in antioxidant enzyme activity and levels of antioxidant compounds (ascorbate and glutathione), the content of reactive oxygen species
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(ROS) (superoxide anion, hydrogen peroxide) and malondialdehyde were higher than in control seedlings. LA significantly promoted osmoregulator level and antioxidant enzyme activities compared to stressed-seedlings alone. Also, it both increased levels of ascorbate and glutathione and regenerated their oxidized forms, thus contributing to maintaining cellular redox status. Similarly, LA prevented excessive accumulation of Na+ and promoted K+/Na+ ratio and Ca content. ROS content was significantly reduced, and the inhibitions in the above parameters markedly recovered. CONCLUSION: LA reduced salinity-induced oxidative damage and thus contributed to growth and development of plants in saline soils by modulating ion homeostasis between plant and soil as well as in osmoregulator content and antioxidant system. Keywords: Lipoic acid, salt stress, wheat, antioxidant system, ion homeostasis, osmoregulator, RuBisCo
INTRODUCTION High salinity is among the most detrimental of abiotic stresses, and it causes serious dysfunction in many vital processes, from the structural changes in membranes to major metabolic reactions such as photosynthesis, protein synthesis, and energy metabolism
1-3
.
Many researchers have described in detail the morphological and metabolic changes arising from high salinity for a number of plant species during all growth and developmental stages, from germination through florescence 4, 5. In addition, these studies revealed that plants have developed many defence strategies to tolerate the harmful effects of exposure to salt stress, including elevating the levels of osmoregulators such as proteins, carbohydrates and proline; activating the enzymatic and non-enzymatic antioxidant systems; and maintaining ion homeostasis and the cellular redox status
6, 7
. However, depending on the severity and
duration of the stress, these strategies are often insufficient to cope with the detrimental effects. Therefore, several methods have been employed to induce salt tolerance in especially economically important plants. One of these methods is to exogenously apply various substances such as plant growth regulators, signal molecules, amino acids, and/or vitamins 8. This article is protected by copyright. All rights reserved.
Lipoic acid (LA, thioctic acid), a vitamin-like compound, was first isolated and chemically identified in 1951 by Reed and colleagues
9, 10
. It is required as a cofactor for the activity of
enzyme complexes involved in the decarboxylation of α-ketoacids and in the glycine cleavage
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system
11, 12
. Moreover, LA, is a power antioxidant molecule with unique properties because
both its own and its reduced form dihydrolipoic acid (DHLA) have reducing property. The low redox potential of the DHLA/LA couple makes it a powerful reductant that reduces glutathione disulfide, vitamin C, vitamin E, and scavenges directly reactive oxygen species (ROS) such as superoxide, singlet oxygen, hydroxyl and peroxyl radicals
13
. Therefore, in
animals many studies have focused the potential of free LA and DHLA as powerful metabolic antioxidants
13-15
. Similarly, LA has been detected in roots and leaves of several plants, and
investigated its effects on recycling of other antioxidants, ROS production, and redox status of cells 16, 17. However, any studies have not been carried out exhibiting effects of exogenous LA application on plants under stressed or unstressed conditions. The objective of this study was to determine the effects of exogenously pre-treating saltstressed wheat seedlings with LA with regard to their morphology (leaf surface area), physiology (relative water content (RWC), inorganic element composition), biochemistry (proteins, carbohydrates, proline, and chlorophyll contents, antioxidant activity, and oxidative stress parameters), and molecular parameters (RuBisCo expression), and thus to contribute to elucidating the effect mechanism of LA in plants. This study is the first to investigate the effects on plants of applying LA exogenously.
MATERIALS AND METHODS
Plant growth and treatments Wheat seeds (Triticum aestivum cv Altındane) were used in the present study. Seeds of wheat were surface-sterilized with 5 % (v/v) sodium hypochlorite for 10 min followed by thorough washing in distilled water. After sterilization, the seeds were soaked in distilled water for about 5 h and then sowed in sand-filled pots. Seedlings were grown in a growth chamber under controlled environmental conditions for 11 days (day/night temperature of 25/20◦C, relative humidity of 75%, and a 16-h photoperiod). Modified Hoagland solution containing 5 mM KNO3, 1 mM NH4NO3, 0.5mM KH2PO4, 5 mM Ca(NO3)2 3 4H2O, 1.5 mM Fe-EDTA, and 2 mM MgSO4 3 7H2O, and other micronutrients in their original concentration were used as the fertilizer. Lipoic acid (LA) and NaCl treatments to wheat seedlings were started after 11 days of sowing. LA solution (1. 75 mM) was sprayed wheat seedlings. For salt stress
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induction, a single dose of NaCl solution (100 mM) was given to each plastic pot. These concentrations of LA and NaCl were selected after preliminary studies. The seedlings treated without NaCl was used as control group. Finally, the plants were harvested in 14th day of
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sowing and it is recorded some changes in physiological, biochemical and molecular parameters.
Determination of relative leaf water content The relative water content (RWC) in leaves was estimated as per the method of Barrs and Weatherly 18. Leaf relative water content was obtained from the following formula: RWC (%) = (fresh weight − dry weight)/ (saturated weight − dry weight) ×100
Determination of soluble protein, proline and total carbohydrates contents Soluble protein content in leaves was measured according to methods of Smith and colleagues 19
. Bovine serum albumin was used as standard and the results were calculated as ‘mg
protein/g FW’. For the estimation of the amount of proline was used method of Bates and colleagues (Bates, Woldren and Teare
20
. To quantify of total carbohydrates, the method
described by Dische was applied with a slight modification 21.
Determination of pigment content and western blotting Chlorophyll content in leaves determined according to methods of Witham and colleagues 22. The results were expressed as mg/g FW. SDS-PAGE was performed as described by Laemmli, and proteins were separated by molecular size
23
. Then, separated proteins according to molecular size determined by using
the method of Towbin and colleagues 24. Transfer operation was carried out in blotting device with a half hour with 60 mA current. After transfer, 1: 2000 diluted anti- RuBisCo antibody (ab62391) and anti-mouse secondary antibodies (diluted 1: 10000) was used. Using a Crossreacted antibody Super Signal West Pico Chemiluminescent Substrate (Pierce, Perbio Science UK), RuBisCo protein bands were detected.
Determination of antioxidant enzyme activities For the determination of activities of antioxidant enzymes, 0.5 gr leaf tissue was homogenized in 5 mL of 0.1 M phosphate buffer (pH 6.75) containing 0.3 % (w/v) polyvinylpyrolidone and 1 mM ethylenediaminetetraacetic acid (EDTA). The homogenate was centrifuged at 12.000×g
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for 15 min at 4 °C and the supernatant was used as the source of enzymes and soluble protein content. The activity of superoxide dismutase (SOD) was assayed by the inhibition of the
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photochemical reduction of nitroblue tetrazolium chloride (NBT)
25
.The guaiacol peroxidase
(GPX) activity was determined in the homogenates by measuring the increase in absorption and color development at 470 nm due to the guaiacol (hydrogen donor) oxidation was recorded for 5 min
26
. The catalase (CAT) activity was measured at 240 nm for H2O2
decomposition rate using the extinction coefficient of 40 mM-1 cm-1
27
. The ascorbate
peroxidase (APX) activity was determined by monitoring the decrease in absorbance at 290 nm as reduced ascorbic acid (AsA) was oxidized (extinction coefficient of 2.8 mM-1 cm-1) 28
.Te glutathione reductase (GR) activity was determined using the method described by
Foyer and Halliwell and assayed by monitoring the decrease in absorbance at 340 nm because of NADPH oxidation 29.
Determination of contents of superoxide anion, hydrogen peroxide and malondialdehyde The production rate of superoxide was determined according to Elstner and Heupel with a slight modification 30. Hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents were measured according to Velikova and colleagues 31.
Determination of the amount of antioxidant compounds The contents of AsA, oxidized ascorbate (dehydroascorbate, DHA), and total ascorbate (AsA+DHA); total glutathione (GSH+GSSG), oxidized glutathione (GSSG), and reduced glutathione (GSH) contents were determined according to Wu and colleagues that was modified from the method of Hodges and colleagues 32, 33.
Preparation of plant samples and ICP/OES experimental procedure The leaves of wheat seedlings were oven-dried at 68˚C for 48 h. The dried samples were ground in the grinding machine. Inorganic element contents (Na+, K+, and Ca) were determined after wet digestion of dried and ground sub-samples using a HNO₃-H₂O₂ acid mixture (2:3 v/v) with three step (first step 145ºC, 75% RF, 5 min; second step 180ºC, 90% RF, 10 min and third step 100ºC, 40% RF, 10 min) in microwave. The content of the elements were determined by inductively couple plasma spectrophotometry (Perkin-Elmer, Optima 2100 DV, ICP/OES, Shelton, CT 06484-4794, USA) 34.
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Statistical analysis The experiment was a completely random design with six replications with three parallel. All data obtained were subjected to a one-way analysis of variance (ANOVA), and the mean
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differences were compared by Duncan’s multiple range test. In all the tables, the spread of values is shown as standard errors of the means.
RESULTS
Effect of exogenous LA on RWC, leaf surface area, chlorophyll content and RuBisCo expression in wheat seedlings under salt stress RWC was adversely affected by salinity. A 11% decline was observed compared to control. LA pretreatment had a positive effect on RWC, and resulted in more than 5.6% amelioration compared to stressed-seedlings (Table 1). As parallel to decrease in RWC, leaf surface area was significantly reduced by salt application. As shown Table 1, exogenous LA remarkably promoted leaf surface area in wheat seedlings with or without the stress. The reduction of 13% in the stressed-seedlings was ameliorated up to 7.6% with LA pretreatment. Significant reduction was observed in chlorophyll content and RubisCo expression of wheat seedlings under salt stress in comparison with the control. In contrast, LA application was effective in turning back inhibitions in these parameters in comparison to stressed-seedlings alone (Table 1, Fig 1). Effect of exogenous LA on Na+, K+ and Ca contents in wheat seedlings under salt stress As shown Table 2, salt stress elevated Na+ content up to 14-fold as compared to control; however, it reduced K+/Na+ ratio from to 41 to 3.5 in spite of an increase in K+ content. Similarly, Ca content was also reduced by salt stress. The stressed-seedlings accumulated significantly lower amount of Na+ and higher amounts of K and Ca upon foliar application of LA as compared to the stressed-seedlings without LA. Eventually, LA application increased K+/Na+ ratio and recovered ion homeostasis.
Effect of exogenous LA on level of osmoregulators in wheat seedlings under salt stress Effects of salt stress and LA application on level of osmoregulators in leaves of wheat were assessed in terms of proline, soluble protein, and total carbohydrate level. Salt stress caused to a rise reaching an 8% greater level over the control in protein level. LA pretreatment resulted This article is protected by copyright. All rights reserved.
in further increase reaching 16.5% compared to stressed-seedlings alone. Under normal conditions also LA application increased slightly protein level compared to the control (Table 1). Similarly, proline and carbohydrate levels also exhibited same trends. Salt-induced
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increases in levels of these solutes were further promoted by LA pretreatment.
Effect of exogenous LA on activities of antioxidant enzymes in wheat seedlings under salt stress As shown Table 3, compared with the control, activities of antioxidant enzymes SOD, GPX, CAT, APX and GR were markedly promoted in the seedlings under saline conditions whether they had been pretreated with LA or not. LA-pretreated seedlings had higher activities (except CAT) compared to stressed-seedlings alone.
Effect of exogenous LA on ascorbate-glutathione pool in wheat seedlings under salt stress A meaningful increase in total AsA and GSH contents were observed with salt treatment (Table 4). LA application further increased contents of these substances. On the other hand, while salt stress significantly elevated levels of DHA and GSSG compared to control, it reduced levels of AsA and GSH. Thus, salinity led to low levels of AsA/DHA and GSH/GSSG ratios. LA application was effective in decreasing the DHA and GSSG contents, and increasing the AsA and GSH contents. Thus, LA application significantly promoted the AsA/DHA and GSH/GSSG ratios. Under unstressed conditions also LA application resulted in a small increase in total AsA and GSH contents.
Effect of exogenous LA on MDA and ROS contents in wheat seedlings under salt stress Prior studies depicted that the plants suffer from oxidative stress and membrane damage under saline conditions, as evidenced by accumulation of ROS and MDA. In this study, to investigate whether the alleviating effect of LA on wheat seedlings suffering from salt stress was related to oxidative stress and membrane damage, we measured superoxide anion, hydrogen peroxide, and MDA contents. Salt stress adversely affected the membrane integrity. The MDA content was elevated by salt stress by 25.6% over to control. Exogenous application of LA resulted in a marked amelioration of deleterious effect of salinity on MDA content. It brought down the MDA content by 12% compared to stressed-seedlings. Similarly, LA application decreased the MDA content (by 4%) compared to control under normal conditions (Table 5). A significant
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increase in superoxide anion and hydrogen peroxide levels was observed at saline conditions. These raises reached by 43% and 53% in the stressed-seedlings, respectively. The saltinduced increase in ROS content was largely abolished by the pretreatment of LA. In
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addition, LA application resulted in a small decrease in these parameters compared to control under normal conditions (Table 5).
DISCUSSION The salt-induced reductions in growth and development are mainly associated with water deficiency, nutrient imbalance and specific ion toxicity
35
. Due to high osmotic pressure in
saline environments, the uptake of water and minerals by roots is getting difficult. This case causes the imbalance between water uptake and transpiration and eventually impairs metabolic activities leading to the reduction in growth and development 36. RWC is one of the most useful indicators of the physiological water status of plants in terms of cellular hydration under the possible effects of leaf water potential and osmotic adjustment 37. The present study showed that salt stress reduced RWC significantly when compared to control seedlings. It is highly possible that the reduction in RWC was linked to higher concentrations of Na+ and Clions in media, which reduce significantly uptake of water by roots due to low osmotic potential. Moreover, the reduction in turgidity of leaves arising from low RWC caused a significant inhibition in leaf surface area, an important variable in crop growth patterns. These results are in harmony with the findings reported from previous researchers, who observed that salt stress decreased water uptake and RWC with a concomitant reduction in leaf area 38, 39
. The reduced RWC both caused a reduction in surface area by decreasing turgidity of leaves
and suppressed stomatal conductance as stated by prior researchers
39, 40
. Nevertheless,
exogenous LA treatment significantly mitigated the salt-induced decrease in RWC and hence contributed to increase leaf surface area. This study revealed that the enhancement in RWC and leaf surface area due to foliar application of LA was due to its modulating role on ion homeostasis, osmotic regulation and antioxidant system or their combination. Ion homeostasis and osmotic regulation play crucial roles in maintaining metabolic processes and cell turgidity 4, 35. Changes in these parameters are important for determining the level of plant stress tolerance. Many researchers informed that salt stress led to the disturbance of ion homeostasis with the excessive accumulation of toxic ions, which causes a great deleterious effect on critical metabolic processes like water status, nutrient uptake and photosynthetic efficiency
41-43
. The findings of present study clearly showed that salt stress increased Na+
content approximately 14-fold in the leaves of wheat seedlings with a concomitant decrease in This article is protected by copyright. All rights reserved.
K+/Na+ ratio. This ratio is used as a good indicator of plant’s tolerance level. K+ has a distinctive role in osmotic regulation as well as in photosynthesis efficiency, protein synthesis, and enzyme activation
44-46
. It is well-documented Na+ ions disturb metabolic
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processes by superseding K+ in chemical reactions. We found that salt stress significantly reduced K+/Na+ ratio from 41-fold to 3.5-fold in spite of an increase in K+ content. This means that wheat seedlings tried to keep K+/Na+ ratio high to survive, but did not achieve it sufficiently. LA application both increased K+ content by 40%, and reduced nearly fifty-fifty excessive accumulation of Na+. In brief, LA mitigated largely salt-induced reduction in K+/Na+ ratio and thus contributed to increase in RWC and ultimately leaf surface area. It is well-documented that under saline conditions, maintaining the cellular membranes’ integrity is an influential integral part of tolerance mechanism 47. We thought that LA carried out this modulating effect by maintaining membrane stability and selectivity. This assumption was confirmed by high Ca content and low membrane damage determined in LA-applied seedlings. LA increased remarkably Ca content compared to control under stressed and unstressed conditions, whereas salt stress reduced markedly Ca content compared to control. Increase in Ca content has critical important in terms of resistance of plants, because Ca can reduce membrane damages by ensuring stability and continuity of membrane structure
48, 49
.
In addition to its protective effect on membranes, Ca has also a pivotal signalling function such as direct inhibitory effect on a Na+ entry system and a facilitative effect on K+ entry system to maintain higher K+/Na+ selectivity in response to salt stress
50, 51
. Briefly, LA
resulted in maintaining membrane stability and high K+/Na+ ratio through Ca, and thus mitigated salt-induced distortions in membranes and metabolic processes. In this study, membrane damage was evaluated through MDA production. The salt-induced increase in MDA content was reported earlier in wheat seedlings
52
. Similarly, in this study
salt stress enhanced markedly MDA content compared to control, whereas LA application turned back significantly this raise. This data was consistent with high content of Ca, which results in maintaining membrane stability. Osmotic adjustment and ion homeostasis are consisting of a combination of different metabolic and physiological processes. Therefore, increase in Ca alone is not adequate maintaining integrity of cellular membranes. This study revealed that salinity, LA and their combination brought about marked changes in level of osmoregulators and antioxidant system of wheat seedlings. Enhanced accumulation of compatible solutes is among the most characteristic responses of plants exposed to salt stress
36, 52
. Since it needs to an energy cost, synthesis of these solutes
causes a potential growth penalty in terms of carbon allocation. However, they may allow the This article is protected by copyright. All rights reserved.
plant to survive at the expense of plant growth under salt stress
1, 53
. Proteins, carbohydrates
and proline are some of major compatible solutes known in plants. These solutes take part actively many critical processes such as osmotic regulation, scavenging of ROS, and
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stabilization of membrane integrity and stability
54-56
. The data for content of these solutes
shows that salt stress enhanced significantly content of soluble protein, carbohydrate and proline compared to control. These results were in agreement with the findings from prior researchers, who observed marked raises in contents of these solutes
3, 36
. Moreover, LA
application resulted in a greater raise than in stressed seedlings alone. In brief, LA has an important stimulative effect on osmoregulators, and thus further promoted the plant response against salinity. Reduction in photosynthesis efficiency is one of the most characteristic damages of salt stress in the plants
57, 58
. A number of researchers have reported that chlorophyll content is notably
reduced by salt stress
35, 52
. It seems likely that this decrease was arising from decrease in
chlorophyll synthesis and increase in chlorophyllase activity. In addition to decrease in chlorophyll content, excessive Na+ accumulation inhibits the activities and expressions of enzymes involved in CO2 assimilation 59. We observed that RubisCo expression significantly was reduced by salt stress. From above data on Na+ content, it is clear that excessive Na+ accumulation caused drastically reduction in RubisCo expression. Also, a combination of decreased RWC, leaf area, stomatal conductance, chlorophyll content, and RubisCo expression led to a limitation in CO2 assimilation and ultimately caused to excess production of ROS in electron transport system (ETS) by decreasing the consumption of ATP and NADPH. Nevertheless, higher levels of RWC, chlorophyll content and RuBisCo expression as compared to stressed-seedlings alone inferred that LA application had a marked mitigating effect on both ETS and CO2 assimilation stages of photosynthesis, and turned back notably reduction in these parameters. This promoting effect on photosynthesis of LA was linked to its combine regulatory effect on ion balance, osmoregulator content, and RWC. In LA-treated seedlings, the marked decrease in ROS content supported strongly above findings. In accordance with high MDA content, salt stress also caused to a significant increase in superoxide anion and hydrogen peroxide levels. In contrast, LA application resulted in a marked decrease in amount of ROS under stressed and unstressed conditions. The reduction in ROS content is a natural result of advance in plant defence mechanism. The data from above indicate that LA contributed to the normalization of metabolic pathways, and eventually reduced ROS production.
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As well as to pathways reducing the production of ROS, the other crucial mechanism is to scavenge the produced ROS. For this purpose, plants had enzymatic and non-enzymatic antioxidant defence system. This study showed that stressed-seedlings had an increase in
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activities of antioxidant enzymes SOD, GPX, CAT, APX, and GR as compared to control group. In spite of the enhanced enzyme activities, the measured high ROS content strongly suggested that this increase was not adequately to cope with the stress. However, LA application resulted in further increases in enzyme activities (except CAT) as compared to stressed-seedlings. In LA-treated seedlings, low ROS content concomitant with high activities of enzymes clearly means that LA contributed to improve the plant resistance against the salinity by scavenging effectively ROS through enhanced antioxidant activity. Also, owing to free radical scavenging properties, LA and its reduced form DHLA might have directly contributed maintaining low levels of them by scavenging ROS. The marked reduction determined in superoxide anion and hydrogen peroxide contents means that LA contributed directly ROS scavenging as well as the antioxidant enzymes did. The prior studies provided evidence that in animals LA application restored reduced levels of the antioxidant substances such as AsA and GSH
13, 60
. Up to date, only a few studies were
reported exhibiting the relationship between endogenous level of LA/DHLA and the other antioxidant substances in plants
16, 17, 61
. We found that salt stress had an important effect on
contents of total AsA and GSH, and levels of their reduced (AsA and GSH) and oxidised (DHA and GSSG) forms. Under salt stress, total AsA, total GSH, DHA and GSSG contents elevated significantly compared to control, but their reduced forms AsA and GSH decreased markedly. However, while LA application both total contents and reduced forms of these compounds further increased as compared to stressed-seedlings, it reduced significantly their oxidised forms. It is known that redox potential of LA/DHLA pair can reduce GSSG and DHA without the involvement of reducing equivalents. In this study, LA-induced high activities of APX and GR suggested that LA resulted in reduction the DHA and GSSG via both enzymatic APX and GR and its own redox property. From all these findings it is clear that LA both regenerated AsA and GSH and increased their total levels, and thus contributed to maintaining cellular redox status and improvement of non-enzymatic antioxidant defence system.
CONCLUSION The study clearly exhibited that under saline conditions exogenous LA application affected positively ion homeostasis, osmoregulator content, and antioxidant system in leaves of wheat This article is protected by copyright. All rights reserved.
seedlings and clearly contributed to response to oxidative damage and metabolic distortions. Although certain aspects of effect mechanism of LA is not clear here, these results will form the basis for further studies leading to a more detailing understanding of involvement of LA in
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the response to stress and be a good model to improve resistance of plants subjected to various environmental stresses.
ACKNOWLEDGMENT This work was supported by a grant from the research funds appropriated to Ataturk University, Erzurum, Turkey (2012-480).
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34. Mertens D, In: Plants Preparation of Laboratory Sample: . AOAC Official Method 922.02, AOAC-International Suite 500, 481. North Frederick Avenue, Gaitherburg, Maryland, USA (2005). 35. Kaya C, Sonmez O, Aydemir S, Ashraf M and Dikilitas M, Exogenous application of mannitol and thiourea regulates plant growth and oxidative stress responses in saltstressed maize (Zea mays L.). J Plant Interact 8:234-241 (2013). 36. Bastam N, Baninasab B and Ghobadi C, Improving salt tolerance by exogenous application of salicylic acid in seedlings of pistachio. Plant Growth Regul 69:275-284 (2013). 37. Gonzalez L and Gonzalez-Vila M, Determination of Relative Water Content. In: Handbook of Plant Ecophysiology Techniques. Springer, Netherlands (2001). 38. Yokafi B, Tuna AL, Burun B, Altunlu H, Altan F and Kaya C, Response of the tomato (Lycopersicon esculentum Mill.) plant to exposure to different salt forms and rates. Turkish J Agr Forest (2008). 39. Ziaf K, Amjad M, Pervez MA, Iqbal Q and Rajwana IA, Evaluation of different growth and physiological traits as indices of salt tolerance in hot pepper (Capsıcum annuum L.). Pakistan J Bot 41:1797-1809 (2009). 40. Lycoskoufis LH, Savvas D and Mavrogianopoulos G, Growth, gas exchange and nutrient status in pepper (Capsicum annum L.) grown in re-circulating nutrient solution as affected by salinity imposed to half of the root system. Scientia Hort 106:147-161 (2005). 41. Habib N and Ashraf M, Effect of exogenously applied nitric oxide on water relations and ionic composition of rice (Oryza sativa L.) plants under salt stress. Pakistan J Bot 46:111-116 (2014). 42. Xue Z, Zhao S, Gao H and Sun S, The salt resistance of wild soybean (Glycine soja Sieb. et Zucc. ZYD 03262) under NaCl stress is mainly determined by Na+ distribution in the plant. Acta Physiol Plant 36:61-70 (2014). 43. Ahmad Z, Tahir S, Ab d, M. and Amanullah M, Salt-Induced Variations in Physiological Parameters and Nutrient Concentrations of Two Wheat Cultivars. Commun Soil Sci Plan 45:29-41 (2014). 44. Peng YH, Zhu YF, Mao YQ, Wang SM, Su WA and Tang ZC, Alkali grass resists salt stress through high [K+] and anendodermis barrier to Na+. J Exp Bot 55:939-949 (2004). 45. Takahashi R, Nishio T, Ichizen N and Takano T, Salt-tolerant reed plants contain lower Na+ and higher K+ than salt-sensitive reed plants. Acta Physiol Plant 29:431-438 (2007). 46. Erdal S and Dumlupinar R, Exogenously treated mammalian sex hormones affects inorganic constituents of plants. BiolTrace Elem Res 143:500-506 (2011). 47. Stevens J, Senaratna T and Sivasithamparam K, Salicylic acid induces salinity tolerance in tomato (Lycopersicon esculentum cv. Roma): associated changes in gas exchange, water relations and membrane stabilisation. Plant Growth Regul 49:77-83 (2006). 48. Cramer GR, Calcium-sodium interactions under salinity stress, in Salinity EnvironmentPlants-Molecules, ed. by Läuchli A and Lüttge U. Kluwer Acad. Publishers pp 205-228 (2002). 49. Dumlupinar R, Genisel M, Erdal S, Korkut T, Taspinar MS and Taskin M, Effects of progesterone, β-estradiol and androsterone on the changes of inorganic element content in barley leaves. Biol Trace Elem Res 143:1740-1745 (2011). 50. Busch DS, Calcium regulation in plant cell and his role in signalling. Annu Rev Plant Physiol 46:95-102 (1995). 51. Erdal S, Comparative Evaluation of the Effects of Bone Powder and Calcium Phosphate on Plant Growth and Development. Phosphorus Sulfur 187:1017-1025 (2012). 52. Erdal S, Alleviation of salt stress in wheat seedlings by mammalian sex hormones. J Sci Food Agr 92:1411-1416 (2012).
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53. Munns R and Tester M, Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651681 (2008). 54. Genisel M, Turk H and Erdal S, Exogenous progesterone application protects chickpea seedlings against chilling-induced oxidative stress. Acta Physiol Plant 35:241-251 (2013). 55. Gill PK, Sharma AD, Singh P and Bhullar SS, Effect of various abiotic stresses on the growth, soluble sugars and water relations of Sorghum seedlings grown in light and darkness. Bulgarian J Plant Physiol 27:72-84 (2001). 56. Aghaleh M, Niknam V, Ebrahimzadeh H and Razavi K, Salt stress effects on growth, pigments, proteins and lipid peroxidation in Salicornia persica and S. europaea. Biol Plantarum 53:243-248 (2009). 57. Poor P, Gemes K, Horvath F, Szepesi A, Simon ML and Tari I, Salicylic acid treatment via the rooting medium interferes with stomatal response, CO2 fixation rate and carbohydrate metabolism in tomato, and decreases harmful effects of subsequent salt stress. Plant Biol 13:105-114 (2010). 58. Ali RM, Elfeky SS and Abbas H, Response of salt-stressed Ricinus communis L: to exogenous application of glycerol and/or aspartic acid. J Biol Sci 8:171-175 (2008). 59. Chaves MM, Flexas J and Pinheiro C, Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Annal Bot 103:551-560 (2009). 60. Packer L and Suzuki YJ, Vitamin E and alpha-lipoate: Role in antioxidant recycling and activation of the NF-kappa B transcription factor. Mol Aspects Med 14:229-239 (1993). 61. Tarchoune I, Sgherri C, Baatour O, Izzo R, Lachaal M, Navari-Izzo F and Querghi Z, Effects of oxidative stress caused by NaCl or Na2SO4 excess on lipoic acid and tocopherols in Genovese and Fine basil (Ocimum basilicum). 163: 23-32 (2013).
Table 1. Effects of foliar application of lipoic acid (LA) on relative water content, leaf surface area, and contents of soluble protein, proline, total carbohydrate and total chlorophyll of 14-day-old wheat seedlings exposed to salt stress. Different letters in the same column indicate statistically significant differences (p
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level and antioxidant system Zeynep GORCEK and Serkan ERDAL* Department of Biology, Science Faculty, Ataturk University, Erzurum, Turkey
* Correspondence Serkan ERDAL Tel: +90 442 231 4206 Fax: +90 442 236 0948 E-mail address: [email protected]
Abstract BACKROUND: Soil salinity is one of the most detrimental environmental factors affecting the growth and limiting the agricultural productivity of plants. This study investigated whether exogenous lipoic acid (LA) pretreatment plays a role in promoting salt tolerance in wheat seedlings. The seedlings were treated with LA (1.75 mM) and salt (100 mM NaCl) separately and their combination. RESULTS: Salt stress significantly reduced relative water content (RWC), leaf surface area, ribulosebisphosphate carboxylase (RuBisCo) expression, and chlorophyll content but increased the content of osmoregulators protein, carbohydrates, and proline. In addition, salinity led to an imbalance in the inorganic composition of wheat leaves.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.7020
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While it elevated Na+ content compared to control, Ca content and K+/Na+ ratio were reduced. Under saline conditions, despite increases in antioxidant enzyme activity and levels of antioxidant compounds (ascorbate and glutathione), the content of reactive oxygen species
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(ROS) (superoxide anion, hydrogen peroxide) and malondialdehyde were higher than in control seedlings. LA significantly promoted osmoregulator level and antioxidant enzyme activities compared to stressed-seedlings alone. Also, it both increased levels of ascorbate and glutathione and regenerated their oxidized forms, thus contributing to maintaining cellular redox status. Similarly, LA prevented excessive accumulation of Na+ and promoted K+/Na+ ratio and Ca content. ROS content was significantly reduced, and the inhibitions in the above parameters markedly recovered. CONCLUSION: LA reduced salinity-induced oxidative damage and thus contributed to growth and development of plants in saline soils by modulating ion homeostasis between plant and soil as well as in osmoregulator content and antioxidant system. Keywords: Lipoic acid, salt stress, wheat, antioxidant system, ion homeostasis, osmoregulator, RuBisCo
INTRODUCTION High salinity is among the most detrimental of abiotic stresses, and it causes serious dysfunction in many vital processes, from the structural changes in membranes to major metabolic reactions such as photosynthesis, protein synthesis, and energy metabolism
1-3
.
Many researchers have described in detail the morphological and metabolic changes arising from high salinity for a number of plant species during all growth and developmental stages, from germination through florescence 4, 5. In addition, these studies revealed that plants have developed many defence strategies to tolerate the harmful effects of exposure to salt stress, including elevating the levels of osmoregulators such as proteins, carbohydrates and proline; activating the enzymatic and non-enzymatic antioxidant systems; and maintaining ion homeostasis and the cellular redox status
6, 7
. However, depending on the severity and
duration of the stress, these strategies are often insufficient to cope with the detrimental effects. Therefore, several methods have been employed to induce salt tolerance in especially economically important plants. One of these methods is to exogenously apply various substances such as plant growth regulators, signal molecules, amino acids, and/or vitamins 8. This article is protected by copyright. All rights reserved.
Lipoic acid (LA, thioctic acid), a vitamin-like compound, was first isolated and chemically identified in 1951 by Reed and colleagues
9, 10
. It is required as a cofactor for the activity of
enzyme complexes involved in the decarboxylation of α-ketoacids and in the glycine cleavage
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system
11, 12
. Moreover, LA, is a power antioxidant molecule with unique properties because
both its own and its reduced form dihydrolipoic acid (DHLA) have reducing property. The low redox potential of the DHLA/LA couple makes it a powerful reductant that reduces glutathione disulfide, vitamin C, vitamin E, and scavenges directly reactive oxygen species (ROS) such as superoxide, singlet oxygen, hydroxyl and peroxyl radicals
13
. Therefore, in
animals many studies have focused the potential of free LA and DHLA as powerful metabolic antioxidants
13-15
. Similarly, LA has been detected in roots and leaves of several plants, and
investigated its effects on recycling of other antioxidants, ROS production, and redox status of cells 16, 17. However, any studies have not been carried out exhibiting effects of exogenous LA application on plants under stressed or unstressed conditions. The objective of this study was to determine the effects of exogenously pre-treating saltstressed wheat seedlings with LA with regard to their morphology (leaf surface area), physiology (relative water content (RWC), inorganic element composition), biochemistry (proteins, carbohydrates, proline, and chlorophyll contents, antioxidant activity, and oxidative stress parameters), and molecular parameters (RuBisCo expression), and thus to contribute to elucidating the effect mechanism of LA in plants. This study is the first to investigate the effects on plants of applying LA exogenously.
MATERIALS AND METHODS
Plant growth and treatments Wheat seeds (Triticum aestivum cv Altındane) were used in the present study. Seeds of wheat were surface-sterilized with 5 % (v/v) sodium hypochlorite for 10 min followed by thorough washing in distilled water. After sterilization, the seeds were soaked in distilled water for about 5 h and then sowed in sand-filled pots. Seedlings were grown in a growth chamber under controlled environmental conditions for 11 days (day/night temperature of 25/20◦C, relative humidity of 75%, and a 16-h photoperiod). Modified Hoagland solution containing 5 mM KNO3, 1 mM NH4NO3, 0.5mM KH2PO4, 5 mM Ca(NO3)2 3 4H2O, 1.5 mM Fe-EDTA, and 2 mM MgSO4 3 7H2O, and other micronutrients in their original concentration were used as the fertilizer. Lipoic acid (LA) and NaCl treatments to wheat seedlings were started after 11 days of sowing. LA solution (1. 75 mM) was sprayed wheat seedlings. For salt stress
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induction, a single dose of NaCl solution (100 mM) was given to each plastic pot. These concentrations of LA and NaCl were selected after preliminary studies. The seedlings treated without NaCl was used as control group. Finally, the plants were harvested in 14th day of
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sowing and it is recorded some changes in physiological, biochemical and molecular parameters.
Determination of relative leaf water content The relative water content (RWC) in leaves was estimated as per the method of Barrs and Weatherly 18. Leaf relative water content was obtained from the following formula: RWC (%) = (fresh weight − dry weight)/ (saturated weight − dry weight) ×100
Determination of soluble protein, proline and total carbohydrates contents Soluble protein content in leaves was measured according to methods of Smith and colleagues 19
. Bovine serum albumin was used as standard and the results were calculated as ‘mg
protein/g FW’. For the estimation of the amount of proline was used method of Bates and colleagues (Bates, Woldren and Teare
20
. To quantify of total carbohydrates, the method
described by Dische was applied with a slight modification 21.
Determination of pigment content and western blotting Chlorophyll content in leaves determined according to methods of Witham and colleagues 22. The results were expressed as mg/g FW. SDS-PAGE was performed as described by Laemmli, and proteins were separated by molecular size
23
. Then, separated proteins according to molecular size determined by using
the method of Towbin and colleagues 24. Transfer operation was carried out in blotting device with a half hour with 60 mA current. After transfer, 1: 2000 diluted anti- RuBisCo antibody (ab62391) and anti-mouse secondary antibodies (diluted 1: 10000) was used. Using a Crossreacted antibody Super Signal West Pico Chemiluminescent Substrate (Pierce, Perbio Science UK), RuBisCo protein bands were detected.
Determination of antioxidant enzyme activities For the determination of activities of antioxidant enzymes, 0.5 gr leaf tissue was homogenized in 5 mL of 0.1 M phosphate buffer (pH 6.75) containing 0.3 % (w/v) polyvinylpyrolidone and 1 mM ethylenediaminetetraacetic acid (EDTA). The homogenate was centrifuged at 12.000×g
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for 15 min at 4 °C and the supernatant was used as the source of enzymes and soluble protein content. The activity of superoxide dismutase (SOD) was assayed by the inhibition of the
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photochemical reduction of nitroblue tetrazolium chloride (NBT)
25
.The guaiacol peroxidase
(GPX) activity was determined in the homogenates by measuring the increase in absorption and color development at 470 nm due to the guaiacol (hydrogen donor) oxidation was recorded for 5 min
26
. The catalase (CAT) activity was measured at 240 nm for H2O2
decomposition rate using the extinction coefficient of 40 mM-1 cm-1
27
. The ascorbate
peroxidase (APX) activity was determined by monitoring the decrease in absorbance at 290 nm as reduced ascorbic acid (AsA) was oxidized (extinction coefficient of 2.8 mM-1 cm-1) 28
.Te glutathione reductase (GR) activity was determined using the method described by
Foyer and Halliwell and assayed by monitoring the decrease in absorbance at 340 nm because of NADPH oxidation 29.
Determination of contents of superoxide anion, hydrogen peroxide and malondialdehyde The production rate of superoxide was determined according to Elstner and Heupel with a slight modification 30. Hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents were measured according to Velikova and colleagues 31.
Determination of the amount of antioxidant compounds The contents of AsA, oxidized ascorbate (dehydroascorbate, DHA), and total ascorbate (AsA+DHA); total glutathione (GSH+GSSG), oxidized glutathione (GSSG), and reduced glutathione (GSH) contents were determined according to Wu and colleagues that was modified from the method of Hodges and colleagues 32, 33.
Preparation of plant samples and ICP/OES experimental procedure The leaves of wheat seedlings were oven-dried at 68˚C for 48 h. The dried samples were ground in the grinding machine. Inorganic element contents (Na+, K+, and Ca) were determined after wet digestion of dried and ground sub-samples using a HNO₃-H₂O₂ acid mixture (2:3 v/v) with three step (first step 145ºC, 75% RF, 5 min; second step 180ºC, 90% RF, 10 min and third step 100ºC, 40% RF, 10 min) in microwave. The content of the elements were determined by inductively couple plasma spectrophotometry (Perkin-Elmer, Optima 2100 DV, ICP/OES, Shelton, CT 06484-4794, USA) 34.
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Statistical analysis The experiment was a completely random design with six replications with three parallel. All data obtained were subjected to a one-way analysis of variance (ANOVA), and the mean
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differences were compared by Duncan’s multiple range test. In all the tables, the spread of values is shown as standard errors of the means.
RESULTS
Effect of exogenous LA on RWC, leaf surface area, chlorophyll content and RuBisCo expression in wheat seedlings under salt stress RWC was adversely affected by salinity. A 11% decline was observed compared to control. LA pretreatment had a positive effect on RWC, and resulted in more than 5.6% amelioration compared to stressed-seedlings (Table 1). As parallel to decrease in RWC, leaf surface area was significantly reduced by salt application. As shown Table 1, exogenous LA remarkably promoted leaf surface area in wheat seedlings with or without the stress. The reduction of 13% in the stressed-seedlings was ameliorated up to 7.6% with LA pretreatment. Significant reduction was observed in chlorophyll content and RubisCo expression of wheat seedlings under salt stress in comparison with the control. In contrast, LA application was effective in turning back inhibitions in these parameters in comparison to stressed-seedlings alone (Table 1, Fig 1). Effect of exogenous LA on Na+, K+ and Ca contents in wheat seedlings under salt stress As shown Table 2, salt stress elevated Na+ content up to 14-fold as compared to control; however, it reduced K+/Na+ ratio from to 41 to 3.5 in spite of an increase in K+ content. Similarly, Ca content was also reduced by salt stress. The stressed-seedlings accumulated significantly lower amount of Na+ and higher amounts of K and Ca upon foliar application of LA as compared to the stressed-seedlings without LA. Eventually, LA application increased K+/Na+ ratio and recovered ion homeostasis.
Effect of exogenous LA on level of osmoregulators in wheat seedlings under salt stress Effects of salt stress and LA application on level of osmoregulators in leaves of wheat were assessed in terms of proline, soluble protein, and total carbohydrate level. Salt stress caused to a rise reaching an 8% greater level over the control in protein level. LA pretreatment resulted This article is protected by copyright. All rights reserved.
in further increase reaching 16.5% compared to stressed-seedlings alone. Under normal conditions also LA application increased slightly protein level compared to the control (Table 1). Similarly, proline and carbohydrate levels also exhibited same trends. Salt-induced
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increases in levels of these solutes were further promoted by LA pretreatment.
Effect of exogenous LA on activities of antioxidant enzymes in wheat seedlings under salt stress As shown Table 3, compared with the control, activities of antioxidant enzymes SOD, GPX, CAT, APX and GR were markedly promoted in the seedlings under saline conditions whether they had been pretreated with LA or not. LA-pretreated seedlings had higher activities (except CAT) compared to stressed-seedlings alone.
Effect of exogenous LA on ascorbate-glutathione pool in wheat seedlings under salt stress A meaningful increase in total AsA and GSH contents were observed with salt treatment (Table 4). LA application further increased contents of these substances. On the other hand, while salt stress significantly elevated levels of DHA and GSSG compared to control, it reduced levels of AsA and GSH. Thus, salinity led to low levels of AsA/DHA and GSH/GSSG ratios. LA application was effective in decreasing the DHA and GSSG contents, and increasing the AsA and GSH contents. Thus, LA application significantly promoted the AsA/DHA and GSH/GSSG ratios. Under unstressed conditions also LA application resulted in a small increase in total AsA and GSH contents.
Effect of exogenous LA on MDA and ROS contents in wheat seedlings under salt stress Prior studies depicted that the plants suffer from oxidative stress and membrane damage under saline conditions, as evidenced by accumulation of ROS and MDA. In this study, to investigate whether the alleviating effect of LA on wheat seedlings suffering from salt stress was related to oxidative stress and membrane damage, we measured superoxide anion, hydrogen peroxide, and MDA contents. Salt stress adversely affected the membrane integrity. The MDA content was elevated by salt stress by 25.6% over to control. Exogenous application of LA resulted in a marked amelioration of deleterious effect of salinity on MDA content. It brought down the MDA content by 12% compared to stressed-seedlings. Similarly, LA application decreased the MDA content (by 4%) compared to control under normal conditions (Table 5). A significant
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increase in superoxide anion and hydrogen peroxide levels was observed at saline conditions. These raises reached by 43% and 53% in the stressed-seedlings, respectively. The saltinduced increase in ROS content was largely abolished by the pretreatment of LA. In
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addition, LA application resulted in a small decrease in these parameters compared to control under normal conditions (Table 5).
DISCUSSION The salt-induced reductions in growth and development are mainly associated with water deficiency, nutrient imbalance and specific ion toxicity
35
. Due to high osmotic pressure in
saline environments, the uptake of water and minerals by roots is getting difficult. This case causes the imbalance between water uptake and transpiration and eventually impairs metabolic activities leading to the reduction in growth and development 36. RWC is one of the most useful indicators of the physiological water status of plants in terms of cellular hydration under the possible effects of leaf water potential and osmotic adjustment 37. The present study showed that salt stress reduced RWC significantly when compared to control seedlings. It is highly possible that the reduction in RWC was linked to higher concentrations of Na+ and Clions in media, which reduce significantly uptake of water by roots due to low osmotic potential. Moreover, the reduction in turgidity of leaves arising from low RWC caused a significant inhibition in leaf surface area, an important variable in crop growth patterns. These results are in harmony with the findings reported from previous researchers, who observed that salt stress decreased water uptake and RWC with a concomitant reduction in leaf area 38, 39
. The reduced RWC both caused a reduction in surface area by decreasing turgidity of leaves
and suppressed stomatal conductance as stated by prior researchers
39, 40
. Nevertheless,
exogenous LA treatment significantly mitigated the salt-induced decrease in RWC and hence contributed to increase leaf surface area. This study revealed that the enhancement in RWC and leaf surface area due to foliar application of LA was due to its modulating role on ion homeostasis, osmotic regulation and antioxidant system or their combination. Ion homeostasis and osmotic regulation play crucial roles in maintaining metabolic processes and cell turgidity 4, 35. Changes in these parameters are important for determining the level of plant stress tolerance. Many researchers informed that salt stress led to the disturbance of ion homeostasis with the excessive accumulation of toxic ions, which causes a great deleterious effect on critical metabolic processes like water status, nutrient uptake and photosynthetic efficiency
41-43
. The findings of present study clearly showed that salt stress increased Na+
content approximately 14-fold in the leaves of wheat seedlings with a concomitant decrease in This article is protected by copyright. All rights reserved.
K+/Na+ ratio. This ratio is used as a good indicator of plant’s tolerance level. K+ has a distinctive role in osmotic regulation as well as in photosynthesis efficiency, protein synthesis, and enzyme activation
44-46
. It is well-documented Na+ ions disturb metabolic
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processes by superseding K+ in chemical reactions. We found that salt stress significantly reduced K+/Na+ ratio from 41-fold to 3.5-fold in spite of an increase in K+ content. This means that wheat seedlings tried to keep K+/Na+ ratio high to survive, but did not achieve it sufficiently. LA application both increased K+ content by 40%, and reduced nearly fifty-fifty excessive accumulation of Na+. In brief, LA mitigated largely salt-induced reduction in K+/Na+ ratio and thus contributed to increase in RWC and ultimately leaf surface area. It is well-documented that under saline conditions, maintaining the cellular membranes’ integrity is an influential integral part of tolerance mechanism 47. We thought that LA carried out this modulating effect by maintaining membrane stability and selectivity. This assumption was confirmed by high Ca content and low membrane damage determined in LA-applied seedlings. LA increased remarkably Ca content compared to control under stressed and unstressed conditions, whereas salt stress reduced markedly Ca content compared to control. Increase in Ca content has critical important in terms of resistance of plants, because Ca can reduce membrane damages by ensuring stability and continuity of membrane structure
48, 49
.
In addition to its protective effect on membranes, Ca has also a pivotal signalling function such as direct inhibitory effect on a Na+ entry system and a facilitative effect on K+ entry system to maintain higher K+/Na+ selectivity in response to salt stress
50, 51
. Briefly, LA
resulted in maintaining membrane stability and high K+/Na+ ratio through Ca, and thus mitigated salt-induced distortions in membranes and metabolic processes. In this study, membrane damage was evaluated through MDA production. The salt-induced increase in MDA content was reported earlier in wheat seedlings
52
. Similarly, in this study
salt stress enhanced markedly MDA content compared to control, whereas LA application turned back significantly this raise. This data was consistent with high content of Ca, which results in maintaining membrane stability. Osmotic adjustment and ion homeostasis are consisting of a combination of different metabolic and physiological processes. Therefore, increase in Ca alone is not adequate maintaining integrity of cellular membranes. This study revealed that salinity, LA and their combination brought about marked changes in level of osmoregulators and antioxidant system of wheat seedlings. Enhanced accumulation of compatible solutes is among the most characteristic responses of plants exposed to salt stress
36, 52
. Since it needs to an energy cost, synthesis of these solutes
causes a potential growth penalty in terms of carbon allocation. However, they may allow the This article is protected by copyright. All rights reserved.
plant to survive at the expense of plant growth under salt stress
1, 53
. Proteins, carbohydrates
and proline are some of major compatible solutes known in plants. These solutes take part actively many critical processes such as osmotic regulation, scavenging of ROS, and
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stabilization of membrane integrity and stability
54-56
. The data for content of these solutes
shows that salt stress enhanced significantly content of soluble protein, carbohydrate and proline compared to control. These results were in agreement with the findings from prior researchers, who observed marked raises in contents of these solutes
3, 36
. Moreover, LA
application resulted in a greater raise than in stressed seedlings alone. In brief, LA has an important stimulative effect on osmoregulators, and thus further promoted the plant response against salinity. Reduction in photosynthesis efficiency is one of the most characteristic damages of salt stress in the plants
57, 58
. A number of researchers have reported that chlorophyll content is notably
reduced by salt stress
35, 52
. It seems likely that this decrease was arising from decrease in
chlorophyll synthesis and increase in chlorophyllase activity. In addition to decrease in chlorophyll content, excessive Na+ accumulation inhibits the activities and expressions of enzymes involved in CO2 assimilation 59. We observed that RubisCo expression significantly was reduced by salt stress. From above data on Na+ content, it is clear that excessive Na+ accumulation caused drastically reduction in RubisCo expression. Also, a combination of decreased RWC, leaf area, stomatal conductance, chlorophyll content, and RubisCo expression led to a limitation in CO2 assimilation and ultimately caused to excess production of ROS in electron transport system (ETS) by decreasing the consumption of ATP and NADPH. Nevertheless, higher levels of RWC, chlorophyll content and RuBisCo expression as compared to stressed-seedlings alone inferred that LA application had a marked mitigating effect on both ETS and CO2 assimilation stages of photosynthesis, and turned back notably reduction in these parameters. This promoting effect on photosynthesis of LA was linked to its combine regulatory effect on ion balance, osmoregulator content, and RWC. In LA-treated seedlings, the marked decrease in ROS content supported strongly above findings. In accordance with high MDA content, salt stress also caused to a significant increase in superoxide anion and hydrogen peroxide levels. In contrast, LA application resulted in a marked decrease in amount of ROS under stressed and unstressed conditions. The reduction in ROS content is a natural result of advance in plant defence mechanism. The data from above indicate that LA contributed to the normalization of metabolic pathways, and eventually reduced ROS production.
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As well as to pathways reducing the production of ROS, the other crucial mechanism is to scavenge the produced ROS. For this purpose, plants had enzymatic and non-enzymatic antioxidant defence system. This study showed that stressed-seedlings had an increase in
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activities of antioxidant enzymes SOD, GPX, CAT, APX, and GR as compared to control group. In spite of the enhanced enzyme activities, the measured high ROS content strongly suggested that this increase was not adequately to cope with the stress. However, LA application resulted in further increases in enzyme activities (except CAT) as compared to stressed-seedlings. In LA-treated seedlings, low ROS content concomitant with high activities of enzymes clearly means that LA contributed to improve the plant resistance against the salinity by scavenging effectively ROS through enhanced antioxidant activity. Also, owing to free radical scavenging properties, LA and its reduced form DHLA might have directly contributed maintaining low levels of them by scavenging ROS. The marked reduction determined in superoxide anion and hydrogen peroxide contents means that LA contributed directly ROS scavenging as well as the antioxidant enzymes did. The prior studies provided evidence that in animals LA application restored reduced levels of the antioxidant substances such as AsA and GSH
13, 60
. Up to date, only a few studies were
reported exhibiting the relationship between endogenous level of LA/DHLA and the other antioxidant substances in plants
16, 17, 61
. We found that salt stress had an important effect on
contents of total AsA and GSH, and levels of their reduced (AsA and GSH) and oxidised (DHA and GSSG) forms. Under salt stress, total AsA, total GSH, DHA and GSSG contents elevated significantly compared to control, but their reduced forms AsA and GSH decreased markedly. However, while LA application both total contents and reduced forms of these compounds further increased as compared to stressed-seedlings, it reduced significantly their oxidised forms. It is known that redox potential of LA/DHLA pair can reduce GSSG and DHA without the involvement of reducing equivalents. In this study, LA-induced high activities of APX and GR suggested that LA resulted in reduction the DHA and GSSG via both enzymatic APX and GR and its own redox property. From all these findings it is clear that LA both regenerated AsA and GSH and increased their total levels, and thus contributed to maintaining cellular redox status and improvement of non-enzymatic antioxidant defence system.
CONCLUSION The study clearly exhibited that under saline conditions exogenous LA application affected positively ion homeostasis, osmoregulator content, and antioxidant system in leaves of wheat This article is protected by copyright. All rights reserved.
seedlings and clearly contributed to response to oxidative damage and metabolic distortions. Although certain aspects of effect mechanism of LA is not clear here, these results will form the basis for further studies leading to a more detailing understanding of involvement of LA in
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the response to stress and be a good model to improve resistance of plants subjected to various environmental stresses.
ACKNOWLEDGMENT This work was supported by a grant from the research funds appropriated to Ataturk University, Erzurum, Turkey (2012-480).
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Table 1. Effects of foliar application of lipoic acid (LA) on relative water content, leaf surface area, and contents of soluble protein, proline, total carbohydrate and total chlorophyll of 14-day-old wheat seedlings exposed to salt stress. Different letters in the same column indicate statistically significant differences (p
