УДК 581.1
Change in Antioxidant Responses against Oxidative Damage in Black Chickpea Following Cold Acclimation1
© 2012 M. Nazari, R. Maali Amiri, F. H. Mehraban, H. Z. Khaneghah
Department of Agronomy and Plant Breeding, University College of Agriculture and Natural Resources of the University of Tehran, Karaj, Iran
Received October 22, 2010
Cold stress is an important factor affecting chickpea (Cicer arietinum L.) plants in winter and early spring. We evaluated the effects of cold stress by measuring lipid peroxidation, membrane permeability, and some enzyme activities involved in the ROS-scavenging system under acclimation and non-acclimation conditions in black chickpea ‘Kaka’, a popular genotype planted, and accession 4322, as a landrace genotype. Under non-acclimation conditions, the genotype 4322 prevented the H2O2 accumulation more efficiently, which led to a decrease in lipid peroxidation and membrane permeability compared to Kaka. Studying the activities of antioxidant enzymes showed that catalase was more effective enzyme in cell protection against H2O2 in 4322 plants. Such response in acclimated plants was more pronounced than in control and non-acclimated plants. In this study, the increase in guaiacol peroxidase and ascorbate peroxidase activities did not preserve cell membranes from oxidative damage in Kaka plants. It was observed that short-term acclimation can induce greater cold tolerance upon the increase of oxidative stress in chickpea plants. This was due to low levels of MDA and electrolyte leakage index, indicating the lower lipid peroxidation and higher membrane stability under the cold stress compared to non-acclimated plants.
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1 This text was submitted by the authors in English.
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Abbreviations: ANOVA - analysis of variance; APX - ascorbate peroxidase; CAT - catalase; ELI - electrolyte leakage index; GPX - guaiacol peroxidase; PPO - polyphenol oxidase.
Corresponding author: Reza Maali Amiri. Department of Agronomy and Plant Breeding, University College of Agriculture and Natural Resources of the University of Tehran, 31587-77871, Karaj, Iran. Fax: 98-261-222-7605; e-mail: rmamiri@ut.ac.ir
Keywords: Cicer arietinum - antioxidant enzymes - cold acclimation - cold stress - electrolyte leakage - lipid peroxidation - oxidative damage

INTRODUCTION
Chilling stress, like other types of abiotic and biotic stresses, induces oxidative processes in plant cells. These processes are initiated by reactive oxygen species (ROS), which arise from disturbed operation of electron transport chains and bring about various manifestations of chilling damage [1]. ROS interact nonspecifically with many cellular components, triggering peroxidative reactions and causing significant damage to essential macromolecules, such as photosynthetic pigments, proteins, nucleic acids, and lipids, and especially damaging the membranes [2]. Cold stress increases membrane permeability characterized by the electrolyte leakage from tissues [3]. Cell membranes also are damaged by ROS, resulting in membrane lipid peroxidation that can be used as a biochemical marker of cold stress in plants [4]. These changes all occur before visual morphological changes manifesting after chilling stress. Therefore, monitoring and controlling of ROS levels is vital for plant cold tolerance. Plants acclimate to environmental low temperature changes by scavenging ROS primarily due to a number of cellular mechanisms that are associated with a vast reprogramming of gene expression and cause the changes in the membrane composition and accumulation of cryoprotectants and antioxidants [5].
Cold-tolerant genotypes have some advantages for chickpea cultivation in Iran under low temperature, because chickpea winter-sowing, when temperatures rarely fall below -10°C even in short-term periods has advantages over traditionally spring-sowing, such as the higher seed yield, water-usage efficiency, and moist growing conditions during relatively mild winter. The main disadvantage of chickpea winter-sowing is the risk of winter-kill. Therefore, there is potential for expanding the range of chickpea winter-sowing by using traditional and nonconventional breeding for improved cold tolerance. The black chickpea plants are believed to be more tolerant to environmental stresses [6], but few studies have been reported on their physiological properties under low-temperature stress. However, cold tolerance is a complex phenomenon, which depends on the nature of cold stress, its interaction with other environmental stresses, and the wide range of plant responses. The study of physiological responses of leaves in accessions (as an indicator of chilling injury) under controlled conditions can be used for genotype evaluation and understanding some mechanisms underlying cold stress. In some places, chickpea is cultivated in winter and experiences cold temperatures from December until February.
In this study, two genotypes of black chickpea were compared in their physiological responses to cold stress under non-acclimation and acclimation conditions.

MATERIALS AND METHODS
Plant material and growth conditions. The study was conducted with two Iranian accessions of black chickpea (Cicer arietinum L.), Kaka and 4322, which are known as a local and a landrace accession, respectively. These accessions were provided by Gene Bank of the Department of Agronomy and Plant Breeding of University of Tehran. Genotype 4322 is distributed in Ardabil province - cold region on Iranian plateau in the north-west of Iran. According to our previous studies, genotype 4322 can be a good candidate for cold tolerance studies with black chickpeas.
After sterilization with 10% sodium hypochlorite for 5 min and soaking in distilled water, seeds of each accession were germinated in Petri dishes at 25°C in darkness and then sown at the rate of 5 seeds per pot. Plants were grown in a growth chamber under the luminescent lamps producing white light of 200 µmol/(m2 s) irradiance, 16/8-h day/night regime, at 25°C and 75% relative humidity for 21 days. Chickpea winter-sowing in Iran, where temperatures rarely fall below -10°C even in short-term periods, has advantages over traditional spring-sowing. Short acclimation is a common event in agricultural systems because most plants are exposed to low above-zero temperature before starting intense cold stress. Therefore, we used such short-term low temperatures in order to assess cold tolerance of accessions. Low temperatures that induce ice formation result in plant death. Low temperature treatment should induce discernible changes in cell membranes without ice formation. The cooling regime adopted in our experiments allowed us to differentiate the examined accessions in terms of their tolerance to low-temperature stress.
Cold stress treatment was started after 3 weeks of seedling growth under optimum conditions. For this treatment, one group of 3-week-old plants was maintained under optimum temperature and other plants were subdivided into the two groups. During experiments, one group of plants was placed into a climatic chamber preliminary chilled to 0°C. The temperature was lowered gradually to -10°C, and plants were incubated at this temperature for 15 min. Leaves were harvested immediately after removing the plants from the cold exposure room for analyses. Another plant group was also treated according to this scheme, but after one day at 10°C used as an acclimation temperature. The cold regime (i.e., the combination of temperature and incubation period) was chosen in preliminary experiments.
Analysis of lipid peroxidation in chickpea leaves. The rate of lipid peroxidation in leaves of chickpea genotypes was determined from the accumulation of MDA [7], the product of secondary peroxidation of lipids. MDA was determined from the color reaction with thiobarbituric acid (TBA) and a subsequent measurement of optical density using a Shimadzu UV-160 spectrophotometer (Japan). To this purpose, the samples of leaves without petioles (300 mg) were selected from the middle part of 3-5 plants. Leaves were homogenized in the extraction buffer (0.1 M Tris-HCl, pH 7.6, containing 0.35 M NaCl). Then 2 ml of 0.5% TBA in 20% trichloroacetic acid were added to 3 ml of the homogenate and incubated on a boiling water bath for 30 min. Samples were centrifuged at 5000 g for 10 min, and optical density of the supernatant was measured at the wavelength of 532 nm with a Shimadzu UV-160 spectrophotometer. The extraction buffer with the reagent served as the control. The concentration of MDA was calculated from molar extinction: C = D/EL, where C is the concentration of MDA (µM); D is the optical density, E is the coefficient of molar extinction (1.56 × 105/(M cm)), and L is the length of the solution layer in the vessel (1 cm). The content of MDA was expressed in µmol/g fr wt [8].
Electrolyte leakage index (ELI). Plants chilling tolerance was assessed by ELI in tissues damaged by cold treatments as described previously [9]. The leaves (80 mg) were incubated at different temperatures, and these samples were washed in distilled water for 5 min to remove electrolytes from the leaflets surface. Then, they were placed in glass tubes, poured with 10 ml of distilled water, and subjected to vacuum infiltration until the disappearance of regions not filled with water. Capped tubes containing samples were placed on a shaker (150 rpm) for 30 min. The water extract containing ions released from tissues was placed in a cell with electrodes, and the electrical conductivity (µS/cm) of the extract was measured using a digital conductivity meter (“Inolab”, Germany) at 25°C. The measurement was repeated after placing the tubes with leaf tissues in a boiling water bath for 10 min followed by shaking for 30 min. The ELI (I, %) was calculated according to the formula: I = [(Lt - L0)/(Lb - L0)] × 100, where Lt is electric conductivity of the sample after cold exposure, L0 is electric conductivity of the sample at temperature of growth, and Lb is an electric conductivity of the same sample after boiling [10]. The ELI represents the leakage of electrolytes from the damaged tissues as the percent of leakage from tissues completely destroyed by boiling (100%). The results were treated statistically using the Paired t-test for pair samples.
Estimation of the H2O2 content. H2O2 content was determined according to Loreto and Velikova method [11]. Leaf fragments (0.35 g) were ground in liquid nitrogen with a mortar and pestle and then homogenized in an ice bath with 5 ml of 0.1% TCA. The homogenate was centrifuged at 12 000 g for 15 min, and 0.5 ml of the supernatant was added to 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 1 ml of 1 M potassium iodide. The absorbance of the supernatant was measured at 390 nm with a Shimadzu UV-160 spectrophotometer. The content of H2O2 was calculated by comparison with a standard calibration curve previously made with different concentration of H2O2 and expressed in µmol/g fr wt.
Enzyme extraction and assay. All extraction procedures were carried out at 4°C, and enzyme activities were determined with a Shimadzu UV-160 spectrophotometer. Total soluble protein content was determined using a commercial Protein Assay (“BioRad”, United States), based on the Bradford method [12]. Samples (0.5 g fr wt) were ground in liquid nitrogen, and enzyme extraction was carried out at 0-4°C. Samples were taken from mid leaflets and homogenized in the extraction buffer (Tris-HCl, pH 7.8) containing 10% glycerol. Extracts were centrifuged at 15 000 g, 4°C for 15 min. The supernatant was used for assaying of catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), and polyphenol oxidase (PPO).
CAT activity. CAT activity was determined by monitoring the initial rate of H2O2 disappearance [13]. The reaction mixture contained 3 ml of phosphate buffer (pH 7.0), 5 µl of 30% H2O2, and 50 µl of crude enzyme extract, and a decrease in absorbance was recorded at 240 nm. CAT activity was expressed in µmol of H2O2 decomposed/(mg protein min).
GPX activity. The reaction mixture consisted of 3 µl of guaiacol and 10 µl of H2O2 (30%) in 3 ml of sodium phosphate buffer, pH 7.0. The 50 µl of crude enzyme preparation was added to 3 ml of the reaction mixture [14]. Changes in absorbance at 470 nm were recorded in 20-s intervals, and the activity of guaiacol peroxidase was expressed in µmol of guaiacol oxidized/(mg protein min).
APX activity. APX activity was measured by recording the decrease in ascorbate content at 290 nm, as ascorbate was oxidized [15]. The assay mixture contained 100 µl of enzyme extract, 600 µl of 0.1 mM EDTA in 1.5 ml of 0.05 M potassium phosphate buffer (pH 7.0), and 400 µl of 0.5 mM ascorbic acid. The reaction started with addition of 400 µl of 30% H2O2, and absorbance was recorded after 3 min. The activity of APX was expressed in µmol of ascorbate oxidized/(mg protein min).
PPO activity. PPO activity was determined by the method of Kar and Mishra with slight modification [16]. The standard reaction mixture consisted of 50 µl of 0.01 M pyrogallol, 3 ml of phosphate buffer (pH 7.0), and the addition of 50 µl of crude enzyme extract initiated the reaction, which was measured spectrophotometrically at 420 nm at 20-s intervals. PPO activity was determined by oxidizing pyrogallol and measuring the absorbance of the reaction products. The results were expressed in µmol of purpurogallin formed/(mg protein min).
Statistical analysis. Recorded data were processed by the analysis of variance (ANOVA) in a factorial experiment on the basis of Randomized Completely Randomized Design (RCRD). The data were analyzed using computer MSTATC software. Main and interaction effects of experimental factors were determined. We presented the results in the form of combination of treatments, and not separately or individually. The means were compared by Duncan’s multiple range tests.

RESULTS
Lipid peroxidation (MDA)
Low-temperature treatment of non-acclimated plants enhanced strongly the MDA content (lipid peroxidation) in Kaka plants, while in plants 4322 it did not have any effect. After 24-h acclimation at 10°C, MDA content declined to near the control level. Thus, 4322 plants were more cold-tolerant than Kaka plants (fig. 1a).

Electrolyte leakage index (ELI)
The effect of low temperature on ELI is shown in fig. 1b. Levels of ELI in both genotypes did not change by stress after acclimation, while treating the plants with subzero temperatures without acclimation provoked an increase in leakage in leaves of 4322 plants and local genotype of chickpea. Also ELI in the 4322 genotype was lower than that in Kaka plants under all conditions.

Changes in the content of H2O2
The effect of cold stress on the content of H2O2 in two genotypes of chickpea, under both acclimated and non-acclimated conditions, is represented in fig. 1c. Compared to control, hydrogen peroxide accumulation increased under non-acclimated conditions in both genotypes. Acclimation of the accession 4322 led to a decrease in the content of H2O2 after stress, which was lower than that in control leaves. However, its content in local genotype Kaka after acclimation was higher than in the 4322 genotype.

CAT activity
CAT activity in these genotypes increased differently (fig. 2a). CAT activity in 4322 plants under control, acclimated, and non-acclimated conditions was dramatically higher than that in the local genotype. Compared to non-acclimated plants, CAT activity of acclimated plants increased, although it was significantly higher in the genotype 4322.

GPX activity
In response to cold stress, a regular increase in the activity of guaiacol peroxidase was shown in both acclimated and non-acclimated local plants (fig. 2c), but GPX activity did not change significantly in 4322 plants under these conditions. Except control plants, in which peroxidase activity was approximately equal in local and 4322 genotypes, this activity in local plants was higher than in both acclimated and non-acclimated 4322 plants.
APX activity
It was observed that APX activity of both genotypes increased under cold stress in non-acclimated plants, although in Kaka plants it was higher (fig. 2b). In acclimated plants, the level of APX activity in Kaka plants increased while in the 4322 genotype it did not change. Also APX activity in Kaka plants was always higher than that in 4322 plants.

PPO activity
This enzyme activity in two genotypes responded differently to cold acclimation. Although leaves of both genotypes under optimum temperature had almost similar PPO activity, differences were remarkable after cold stress. Accession 4322 had greater PPO activity after stress in both acclimated and non-acclimated plants. No significant difference was detected in PPO activity in both genotypes between cold acclimated and non-acclimated plants (fig. 2d).

DISCUSSION
We evaluated effects of short-term chilling on lipid peroxidation, membrane permeability, and oxidative stress in two black chickpea genotypes, Kaka and 4322. We observed a dramatic cold-induced increase in the MDA level and ROS accumulation in the non-acclimated Kaka genotype. The ELI was used to assess the magnitude of injury to the cell membrane after exposure to short-term low temperature. The higher ELI of the Kaka genotype after cold stress indicates the lower stability of its cell membrane, which may be related to the enhanced lipid peroxidation caused by ROS and changes in membrane lipid compositions that influence membrane permeability [17].
The content of H2O2 changes in response to cold treatment are very important because H2O2 can promote the formation of hydroxyl radical, another and more toxic ROS [18]. We demonstrated that, cold treatment of non-acclimated Kaka plants resulted in H2O2 accumulation that probably was one of the reasons for the increase in the MDA level. Moreover, H2O2 has a signaling role in chilling perception and could motivate some confrontation metabolisms [19]. In order to study this assumption, we measured the activities of antioxidant enzymes CAT, GPX, APX, and PPO. These enzymes are implicated in the ROS detoxification and plant resistance to the cold-induced oxidative stress [20]. Although the H2O2 level in the leaves could be a signal for antioxidant responses but, in the first place, high H2O2 accumulation indicates the lack of antioxidant activity. In the case of cold-sensitive plants, CAT is more implicated than other enzymes [2119].
In our study, CAT activity in Kaka plants did not change significantly under cold stress. Peroxidases as enzymatic antioxidants are thought to be essential for ROS detoxification during normal metabolism and particularly during stress. In the Kaka genotype, GPX and APX activities had similar patterns. The increase in peroxidase activity could be a sign that plants responded to ROS accumulation. But in spite of increasing activity of GPX and APX compared to CAT in Kaka plants, an accumulation of H2O2 and consequently lipid peroxidation occurred. Thus, it seems likely that defense mechanisms in the Kaka genotype could not cope with cold stress damages.
In the 4322 genotype, we observed only slight increments in the MDA content and ELI, which indicates the higher membrane stability in non-acclimated plants, however, the level of H2O2 in leaves increased. Small changes in MDA and ELI are probably a sign that this level of H2O2 could not be considered as an intense damage. Also the H2O2 level in tissues is different for various plants [22убрали]. In this study, the H2O2 level in the 4322 genotype was higher than in Kaka plants under optimum conditions. Moreover, determination of enzyme activities in this genotype showed that in non-acclimated plants induction of CAT in comparison with GPX, APX, and PPO is more important in cell protecting from oxidative damage and is therefore a main factor in the early response to cold stress. Considering the sudden changes of temperature in some regions and the importance of rapid plant responses to these conditions, we conducted experiments with short-term acclimation. Short-term acclimation is a common event experiences by most plants before environmental temperature reaches to low above-zero temperature or freezing.
In acclimated plants, the content of MDA was approximately similar in the two genotypes. This indicates that sensitive plants may also increase capacity for adaptation to low temperatures due to diverse physiological and biochemical mechanisms functioning during acclimation, but the degree of their adaptation could be different and in 4322 plants the level of MDA was slightly less than in Kaka plants. Furthermore, cold acclimation reduced the ELI and improved the membrane stability as compared with non-acclimated plants of both genotypes. A decrease in membrane stability of Kaka plants reflects apparently the extent of lipid peroxidation caused by ROS. However, 4322 plants always had the lower damage index, which could be due to effective scavenging of oxidant molecules. The H2O2 level measurement verified this hypothesis: after short acclimation a dramatic decrease in the H2O2 happened in 4322 plants. Regarding to these results, it is probable that the high activity of antioxidant enzymes in the tolerant genotype (4322) suppresses H2O2 accumulation and lipid peroxidation, thus weakening membrane injury. Short acclimation before cold stress induced a dramatic increase in CAT activity in 4322 plants; in Kaka plants, CAT activity changes were insignificant. Thus, after cold treatment, Kaka plants, which are less resistant to low temperature, showed no change in CAT activity, while in the more resistant plants (4322), the activity of this enzyme significantly increased. These data suggest that CAT activity may directly correlate with the degree of cold tolerance. This result agrees with previous data that CAT is the first enzyme that takes part in H2O2 detoxification [20] and an increase in its activity may decrease the level of H2O2, especially under acclimation conditions. It was reported that under cold stress GPX activity was significantly higher in tolerant genotypes than in sensitive plants [13].
However, in this study we did not observe any association of GPX with cold tolerance. It should be noted that, in our experiments in both cold-susceptible and in cold-tolerant accessions, CAT and peroxidase activities behaved differently. In cold-tolerant accession, early response to short-term low temperature may be related to CAT activation, whereas APX activity was not changed. But at longer cold stress it may be that other enzymes cooperate or replace CAT activity. These results confirm the findings of Willekens et al. [2320] demonstrating opposite changes in CAT and peroxidase activities: plants with higher CAT activity had lower APX activity. Thus, it seems likely that peroxidase activity may be involved in the cold tolerance of chickpea genotypes, but it may be not correlated with the degree of cold tolerance after short acclimation. In this study, no significant changes were seen in PPO activity of two genotypes after cold acclimation. But little increase in PPO activity after acclimation may show that PPO activity needs more time for acclimation.
In conclusion, the black chickpea seedlings showed the capacity of cold tolerance increase when exposed to short acclimation. Some antioxidant enzymes may be activated by such acclimation and this can make chickpea plants ready to encounter lower temperatures. In Kaka plants, cold treatment of non-acclimated and acclimated plants caused a significant increase in GPX and APX activities and did not affect PPO and CAT activities. However, this treatment elevated H2O2 content and therefore stimulated lipid peroxidation and electrolyte leakage. In 4322 plants, such conditions significantly increased CAT and PPO, but not GPX and APX activities, which led to a significant decrease in H2O2 content and stability of cell membranes as compared to Kaka plants. Therefore, different activities of the H2O2-scavenging enzymes in cold-susceptible (Kaka) and cold-tolerant (4322) plants resulted in different intensity of lipid peroxidation in acclimated and non-acclimated plants. In this study, it was observed that 24-h plant exposure to 10°C can trigger necessary mechanisms for cold acclimation in cold-tolerant plants more efficiently than in cold-susceptible plants (Kaka). Therefore, short acclimation can provoke defense systems and alleviate cold damage in 4322 plants. From the results obtained, it may deduced that CAT was more effective enzyme in cell protection against H2O2 under cold stress in cold-tolerant 4322 plants and such protection systems in acclimated plants were more active compared to control and non-acclimated plants. It means that early response to short-term low temperature in 4322 plants may be induced by CAT but not peroxidase. However, under longer cold stress other enzymes may cooperate or replace CAT activity. Thus, it is of interest to study other elements of the defense system and obtain information on expression levels of the related genes functioning under short-term and long-term cold stress; this helps to understand plant strategies under cold stress. Since chickpea plants are cultivated in Iran under dryland conditions, they face drought stress after finishing cold season. The Desi type of chickpea is believed to be more tolerant to drought stress [6]. Therefore, the cold-tolerant genotype 4322, which is rapidly acclimated and survives winter cold can be a good candidate as an alternative plant in high and drought regions, where kabuli chickpeas do not have successful performance. It seems likely that these plants may be used for extending the growing season and geographical range of black chickpea.
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FIGURE CAPTIONS

Fig. 1. Effects of low temperature on MDA content (a), electrolyte leakage index (ELI) (b), and H2O2 content (c) in the leaves of two black chickpea genotypes grown under acclimation and non-acclimation conditions.
1 - accession Kaka, 2 - accession 4322. Different letters above columns represent statistically significant differences based on Duncan’s multiple range tests.

Fig. 2. Antioxidant enzyme activities in the leaves of two Desi-type acclimated or non-acclimated chickpea genotypes subjected to cold treatment.
a - catalase; b - ascorbate peroxidase; c - guaiacol peroxidase; d - polyphenol oxidase. 1 - accession Kaka, 2 - accession 4322. Different letters above the columns represent statistically significant differences based on Duncan’s multiple range tests.