ISSN 10214437, Russian Journal of Plant Physiology, 2012, Vol. 59, No. 6, pp. 708–716. © Pleiades Publishing, Ltd., 2012. .
RESEARCH PAPERS
Some Physiological Responses of Chickpea Cultivars to Arbuscular Mycorrhiza under Drought Stress1 Y. Sohrabia, G. Heidaria, W.Weisanya, K. GhasemiGolezanib, and K. Mohammadic a
Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Kurdistan University, Sanandaj, Iran; fax: +988716620553, email:
[email protected] b Department of Agronomy and Plant Breeding, Faculty of Agriculture, Tabriz University, Iran c Department of Agronomy, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran Received June 19, 2011
Abstract—A factorial experiment based on RCB design with three replicates was conducted to investigate changes in some physiological responses of two chickpea (Cicer arietinum L.) cultivars (Pirouz from Desi type and ILC482 from Kabuli type) to arbuscular mycorrhiza (Glomus etunicatum Becker and Gerdman) under dif ferent irrigation treatments. The experiment was carried out in the greenhouse of the Agricultural Faculty of Kurdistan University from April to August 2009. The results showed that leaf chlorophyll content of chickpea cultivars was significantly increased by arbuscular mycorrhiza (AM) under both well and limited irrigation conditions. Proline accumulation in chickpea leaves under moderate and severe drought stresses was signifi cantly stronger than that under optimum irrigation. Inoculation of chickpea with mycorrhizal fungi caused an increase in the activities of polyphenol oxidase and peroxidase, but a decrease in the activity of catalase. Comparisons among different irrigation levels showed that chickpea plants under drought stress had the most active lipid peroxidation. NonAM plants showed stronger lipid peroxidation under moderate and severe water stresses than AM plants. Lipid peroxidation was more active in Pirouz leaves than in ILC482 leaves. It seems that Kabulitype cultivar responded better to mycorrhizal symbiosis under drought stress than Desi type cultivar. Keywords: Cicer arietinum, arbuscular mycorrhiza, chlorophyll content, drought stress, proline DOI: 10.1134/S1021443712060143 1
INTRODUCTION Chickpea is the third most important grain legume in the world [1]. Chickpea is largely grown under rain fed conditions in arid and semiarid areas, where ter minal drought stress is a major limiting factor. Drought stress has several effects on plants. The response of plants to water stress depends on several factors, such as developmental stage, stress severity and duration, and genotype. Common plant symp toms of water deficit are stunted growth, limited CO2 diffusion to chloroplasts because of stomatal closure, reduced photosynthesis rate, and accelerated leaf senescence [2]. One of the biochemical changes occurring when plants are subjected to these harmful stress conditions is the accumulation of ROS, such as •– superoxide radical ( O 2 ), hydrogen peroxide (H2O2), hydroxyl radical (OH•), and singlet oxygen (1O−), 1 This text was submitted by the authors in English.
Abbreviations: AM—arbuscular mycorrhiza; APX—ascorbate peroxidase; CAT—catalase; MWS—moderate water stress; NAM—nonmycorrhizal plant; POD—peroxidase; PPO— polyphenol oxidase; SWS—severe water stress; WWC—well watering control.
which are inevitable byproducts of normal cell metabolism [3]. These cytotoxic oxygen species are highly reactive, and, in the absence of any protective mechanism, they can seriously disrupt normal metab olism through oxidative damage resulting in lipid per oxidation and consequently membrane injury, protein degradation, enzyme inactivation, pigment bleaching, and disruption of DNA strands [4]. Plants have the antioxidant system to scavenge either ROS or second ary reaction products. The antioxidant system includ ing antioxidant enzymes, such as superoxide dismu tase (SOD), peroxidase (POD), and catalase (CAT), as well as other compounds, such as carotenoids and ascorbate, is the principal defense against oxidants [5]. Plants also produce large amounts of amino acids, such as proline, for improving drought resistance. Pro line prevents oxidation inside the cells under drought stress [6]. In addition to the intrinsic plant stresspro tective systems, plants grow in association with a num ber of soil microorganisms that can alleviate the stress symptoms. Arbuscular mycorrhiza (AM) symbiosis is the most important mutualistic association between AM from soil and plant roots [7]. AM symbiosis con tributes to enhance growth and vigor of plants and can alter plant water relations, particularly during water
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Table 1. Some physical and chemical properties of soil used in the experiment Content, mg/kg soil Texture Sandy loam
pH 6.8
ECe, dS/m 0.73
K
P
Mg
Zn
Mn
Fe
Cu
500
9.50
228.13
1.34
37.30
10.84
1.10
stress periods. Porcel et al. [8] have put forward a hypothesis, according to which AM protected host plants against oxidative damage due to the activation of enzymatic antioxidants. However, the relationship between AM and antioxidants is poorly known. Although antioxidants in bean plants [9] inoculated with AM have been investigated, the changes in chick pea antioxidant enzyme activities in response to drought and AM have not been reported. Therefore, this research was carried out to evaluate the effects of Glomus etunicatum on antioxidant activ ity and some other physiological traits of two chickpea types (Desi and Kabuli) under well and limited irriga tion conditions. MATERIALS AND METHODS Plant material and treatments. An experiment was conducted in the greenhouse of the Agricultural Fac ulty of Kurdistan University from 8 April to 27 August 2009. Some soil physical and chemical properties are presented in Table 1. The soil samples were airdried and crushed to pass through a 2mm sieve. The certi fied seeds of chickpea (Cicer arietinum L.) cultivars were obtained from Agricultural Research Center of Kermanshah, Iran. These seeds were surfacesteril ized with 0.1% HgCl2 for 50 min and washed thor oughly five times with distilled water. The experiment was carried out using a factorial arrangement based on completely randomized design with three replications. Treatments were: two chickpea cultivars (cv. Pirouz of Desi type and cv. ILC482 of Kabuli type) and three irri gation treatments including wellwatering control (WWC), moderate water stress (MWS), and severe water stress (SWS). Plants were inoculated by arbuscu lar mycorrhiza (AM), and nonmycorrhizal (NAM) plants were considered as control. The Glomus etunicatum Becker and Gerdman (AM) was obtained from the Faculty of Agriculture, University of Tabriz, Iran. The AM fungal inoculum consisted of a mixture of rhizospheric soil from trap cultures (Zea mays sp.) containing spores, hyphae, and mycorrhizal root fragments. The inoculated dos age was 10 g of inoculums per 1 kg of soil, each pot containing approximately 790 spores. Chickpea roots were tested for mycorrhizal colonization at 30 days after sowing. Plants were kept under the natural light of day with supplementary light in order to keep a 13h photoperiod with an irradiance at the plant level of 900–11200 μmol/(m2 s) PAR. Temperature and rela tive humidity were 27 ± 3°С and 60 ± 5%, respectively. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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Drought treatments were applied when plants were threeweekold. Chickpea seeds were sown in 20L pots filled with 20 kg of inoculated or not inoculated soil. Irrigation treatments were: control (WWC) plants were watered daily to maintain Ψ not lower than –0.03 MPa; MWS was created by withholding water until a Ψ of ca. –0.6 MPa; SWS was created by withholding water until a Ψ of ca. –1.2 MPa. These water potentials were measured with a WP4 Dew Point Potentiameter (Decagon Devices, Pullman, United States) at the sampling days and were maintained until harvest. To control the water levels, water content in pots was measured daily during the entire experiment with Time Domain Reflectometry TDR method. Due to its flexibility, accuracy, and the possibility for auto mated measurement of several probes simultaneously, time domain reflectometry is a widely applied tech nique for the nondestructive measurement of soil water content [10]. Leaf photosynthetic pigments. For chlorophyll determination, the young leaves were detached from plants after drought treatment. Chlorophyll content in leaves was measured at 50 days (pod initiation) after the onset of the experiment. Prior to extraction, fresh leaf samples were cleaned with deionized water to remove any surface contamination. Fresh leaf samples (0.5 g) were ground in 80% acetone at 4°C with a mor tar and pestle. The absorbance was measured using a UV/visible Shimadzu 160 A spectrophotometer, and chlorophyll content were calculated using the equa tion proposed by Harbone [11]. Total carotenoid content was calculated using the following formula: (1000 × A470) – (3.27 × Ca) – (104 × Cb)/229, where Ca = (12.21 × A663) – (2.81 × A646) and Cb = (20.13 × A646) – (5.03 × A663). Ca and Cb are the concentrations of chlorophylls a and b in mg/g fr wt [12]. Soluble protein. Protein content was determined according to Bradford [13] with BSA as a standard. Proline content. Free proline content in leaves was determined in accordance with the method of Bates et al. [14]. Proline content in leaves was measured at 50 days after the onset of the experiment. Leaf samples (0.5 g) were homogenized in 5 mL of sulfosalicylic acid (3%) with a mortar and pestle. 2 mL of the extract was taken in the test tube, and 2 mL of glacial acetic acid and 2 mL of the ninhydrin reagent were added to it. The reaction mixture was boiled in water bath at 100°C for 30 min. After cooling 6 mL of toluene was No. 6
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added to the reaction mixture, and then it was trans ferred to a separating funnel. After thorough mixing, the chromophorecontaining toluene was separated and absorbance was read at 520 nm with a Camspec M330 UV/Vis spectrophotometer against a toluene blank. Content of proline was estimated using the cal ibration curve built with standard proline solutions. Enzyme assays. About four weeks after drought stress application, young leaves were collected and enzyme activities were assayed. Leaf samples were col lected in an ice bucket and brought to the laboratory. Leaves were then washed with distilled water, and sur face moisture was wiped out. Leaf samples (0.5 g) were homogenized in icecold 0.1 M phosphate buffer (pH 7.5) containing 0.5 mM EDTA with a prechilled mortar and pestle. The homogenate was transferred to centrifuge tubes and centrifuged at 4°C in Beckman refrigerated centrifuge at 15 000 rpm for 15 min. The supernatant was transferred to 30mL tubes and referred to as enzyme extract. Catalase (CAT, EC 1.11.1.6) activity was measured according to Beers and Sizer [15] with minor modifica tions. The reaction mixture (1.5 mL) consisted of 100 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 20 mM H2O2, and 20 μL of the enzyme extract. The reaction was started by the extract addition. A decrease in the H2O2 content was monitored at 240 nm, quantified using its molar extinction coefficient (ε = 36/(M cm)), and the results expressed as U/(mg protein min). Peroxidase (POD, EC 1.11.1.7) activity was esti mated according to Hemeda and Klein [16]. The reac tion mixture contained 25 mM phosphate buffer (pH 7.0), 0.05% guaiacol, 10 mM H2O2, and the enzyme extract. Activity was determined by the increase in absorbance at 470 nm due to guaiacol oxi dation (ε = 26.6/(mM cm). Polyphenol oxidase (PPO, EC1.10.3.1) activity was assayed by the method of Kumar and Khan [17]. The assay mixture contained 2 mL of 0.1 M phosphate buffer (pH 6.0), 1 mL of 0.1 M catechol, and 0.5 mL of the enzyme extract. The mixture was incubated for 5 min at 25°C, after which the reaction was stopped by the adding 1 mL of 2.5 N H2SO4. The absorbance of the purpurogallin formed was read at 495 nm. To the blank, 2.5 N H2SO4 was added to the same assay mix ture at the zero time. PPO activity was expressed in U/(mg protein min). Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined according to Nakano and Asada [18]. The reaction mixture contained 50 mM potassium phosphate (pH 7.0), 0.2 mM EDTA, 0.5 mM ascorbic acid, 2% H2O2, and 0.1 mL of the enzyme extract in a final volume of 3 mL. A decrease in absorbance at 290 nm for 1 min was recorded and the amount of ascorbate oxidized was calculated using extinction coefficient (ε = 2.8/(mM cm)). APX was defined as 1 mmol ascorbate oxidized/mL per min at 25°C.
Determination of the lipid peroxidation rate. Oxida tive damage to leaf lipids, resulting from drought stress, was estimated by the content of total 2thiobar bituric acidreactive substances (TBARS) expressed as equivalents of MDA. TBARS content was estimated by the method of Cakmak and Horst [19] with some modifications. Fresh leaf samples were collected at 50 days after the onset of the experiment, and leaf samples (0.2 g) were ground in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) at 4°C. Following the cen trifugation at 12000 g for 5 min, an aliquot of 1 mL from the supernatant was added to 4 mL of 0.5% (w/v) thiobarbituric acid (TBA) in 20% (w/v) TCA. Samples were heated at 90°C for 30 min. Thereafter, the reac tion was stopped in the ice bath. Centrifugation was performed at 10000 g for 5 min, the absorbance of the supernatant was recorded at 532 nm with a Camspec M330 UV/Vis spectrophotometer and corrected for nonspecific turbidity by subtracting the absorbance at 600 nm. The following formula was applied to calcu late MDA content: MDA (nmol/g fr wt) = [(A532 − A600)V × 1000/ε]/W, where ε is the specific extinction coefficient (155/(mM cm)), V is the volume of crushing medium, and W is the fresh weight of leaf sample. Statistical analysis. Analysis of variance was per formed using the SAS software (v. 9.1). Means were compared, using the Duncan test at the 5% probability level. RESULTS Both AM and NAM plants had significant differ ences in the leaf chlorophyll a content (Table 2). Under MWS and SWS conditions, cv. Pirouz plants having AM had significantly more chlorophyll a than plants lacking AM. In this cultivar, under well irriga tion (WWC) there was no significant difference between chickpea plants with and without mycorrhiza. In ILC482 cultivar under WWC and SWS conditions, AM plants had more chlorophyll a than NAM plants, although there was no significant difference between AM and NAM plants under MWS. Comparing between NAM plants of two cultivars showed that cv. ILC482 produced significantly more chlorophyll a than cv. Pirouz under MWS and SWS conditions. However, under WWC condition, cv. Pirouz had noticeably more chlorophyll a than cv. ILC482. In AM plants, comparison of two cultivars showed that under MWS, cv. Pirouz produced significantly more chloro phyll a than cv. ILC482. On the other hand, under WWC and SWS there was no significant difference between two cultivars (Table 2). The results showed that the leaves of plants having AM produced signifi cantly more chlorophyll a and total chlorophyll in comparison to those without mycorrhiza (Table 3). In the leaves of NAM plants, there were signifi cantly more carotenoids as compared with AM plants
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Table 2. Amounts of chlorophyll a (Chl a) and catalase (CAT) activity in leaves of two chickpea cultivars (Pirouz and ILC482) subjected to different irrigation treatments with (AM) and without (NAM) mycorrhiza application Chl a content, mg/g fr wt
CAT activity, U/(mg protein min)
Treatments Pirouz WWC MWS SWS
0.865 ± 0.024c 1.159 ± 0.363abc 0.652 ± 0.034e 1.641 ± 0.255a 0.459 ± 0.045g 1.225 ± 0.454abc
NAM AM NAM AM NAM AM
Pirouz
ILC482
13.530 ± 0.372bcd 3.408 ± 1.449d 9.028 ± 0.474cd 9.311 ± 5.773cd 13.378 ± 0.702bcd 18.679 ± 7.526bc
7.032 ± 0.369cd 3.258 ± 1.281d 5.334 ± 0.280d 3.638 ± 0.914d 42.489 ± 2.230a 22.566 ± 9.210b
ILC482 0.580 ± 0.037f 1.564 ± 0.436ab 0.835 ± 0.043c 0.993 ± 0.240bc 0.714 ± 0.030d 1.434 ± 0.526ab
Note: Each value is the mean ± SE of three replicates (Duncan’s test, P ≤ 0.05).
Table 3. Orthogonal comparison for amounts of chlorophyll a (Chl a), total chlorophyll (Tchl), carotenoid content, and proline content in chickpea leaves in plants with (AM) and without (NAM) mycorrhiza application Chlorophyll content, mg/g fr wt Mycorrhiza status NAM AM WS Cultivar AM WS × cultivar WS × AM Cultivar × AM WS × cultivar × AM
Chl a
Tchl
0.684 ± 0.036b 1.336 ± 0.145a
1.254 ± 0.066b 1.788 ± 0.175a Significance NS NS ** NS NS NS NS
NS NS ** NS NS NS *
Carotenoid content, mg/g fr wt
Proline content, mg/g dry wt
0.741 ± 0.031a 0.429 ± 0.033b
12.532 ± 0.886a 13.258 ± 1.484a
NS NS ** * NS NS NS
** NS NS NS NS NS NS
Notes: Each value is the mean ± SE of three replicates. Different letters followed values within columns designate significant differences (Duncan’s test, P ≤ 0.05). *, ** Significant at P < 0.05 and P < 0.01, respectively. WS − water stress; NS − not significant.
(Table 3). Interaction of chickpea cultivars and irriga tion levels indicated that there was no significant dif ference between Pirouz and ILC482 in the leaf caro tenoid content under different irrigation treatments. The results also showed that this trait did not signifi cantly differ between irrigation treatments (Table 4). There was a significant difference in leaf soluble protein content between cv. Pirouz and cv. ILC482 only under severe drought stress. The leaf soluble protein content in cv. ILC482 under severe stress was signifi cantly higher than that in cv. Pirouz. Although in most cases, there was no significant difference between well irrigated and stressed plants, but in general the higher soluble protein content was obtained for plants under drought stress (Table 4). Under MWS and nonstress (WWC) conditions, there was a significant difference in leaf soluble protein content between plants inoculated with AM and those lacking AM. Thus, in MWS treatment the plants lack ing mycorrhiza and in optimum irrigation condition RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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the plants having AM had more leaf soluble protein. In plants under SWS, there was no significant difference between chickpea plants with (AM) and without (NAM) mycorrhiza (although plants having mycor rhiza produced some more soluble protein) (Table 5). Drought stress induced proline accumulation in chickpea plants. Thus, proline content under both moderate and severe stress conditions was significantly higher than that under normal irrigation. However, there was no significant difference between proline accumulation by plants under MWS and SWS (Fig. 1, Table 3). Irrigation treatments had no significant effect on polyphenol oxidase (PPO) activity in NAM plants at pod initiation. However, PPO activity of AM plants was significantly reduced under MWS. PPO activity of AM plants was higher than that of nonAM plants under all irrigation treatments (Table 6). Under well irrigation and severe drought stress, inoculation of plants with G. etunicatum caused a sig nificant increase in APX activity (Table 6). Although, No. 6
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Table 4. Amounts of carotenoids and soluble protein in the leaves of two cultivars of chickpea (Pirouz and ILC482) subjected to different irrigation treatments Carotenoid content, mg/g fr wt
Soluble protein content, mg/g fr wt
Irrigation level WWC MWS SWS
Pirouz
ILC482
0.64 ± 0.09a 0.55 ± 0.07a 0.49 ± 0.03a
0.50 ± 0.07a 0.62 ± 0.12a 0.64 ± 0.07a
Pirouz
ILC482
1.18 ± 0.07b 1.30 ± 1.62ab 1.14 ± 0.11b
1.20 ± 0.05b 1.30 ± 0.09ab 1.45 ± 0.10a
Notes: Each value is the mean ± SE of three replicates. Different letters followed values designate significant differences (Duncan’s test, P ≤ 0.05).
Table 5. Amounts of soluble protein, polyphenol oxidase (PPO) and ascorbate peroxidase (APX) activities, and MDA content in the leaves of chickpea subjected to different irrigation treatments with (AM) and without (NAM) mycorrhiza application Irrigation levels WWC MWS SWS
WS Cultivar AM WS × cultivar WS × AM Cultivar × AM WS × cultivar × AM
Soluble protein content, mg/g fr wt NAM
AM
1.03 ± 0.03c 1.48 ± 0.05a 1.20 ± 0.12bc
1.36 ± 0.05ab 1.22 ± 0.10bc 1.39 ± 0.09ab ns ns ns * ** ns ns
PPO activity, APX activity, mmol ascorbate U/(mg protein min) oxidized/(mg protein min) NAM
AM
7.81 ± 23.8 ± 1.20bc 5.10a 6.40 ± 9.66 ± 0.70c 1.50b 6.75 ± 21.50 ± 1.00c 2.90a Significance ns ns ** ns * ns ns
MDA content, nmol/g fr wt
NAM
AM
NAM
AM
15.50 ± 3.67c 78.70 ± 33.00ab 37.00 ± 14.60bc
85.30 ± 41.40ab 55.40 ± 21.00abc 116.90 ± 27.30a
3.77 ± 0.60b 4.94 ± 0.30a 3.59 ± 0.60b
5.05 ± 0.35a 3.86 ± 0.64b 2.24 ± 0.11c
ns ** * ns * ns ns
** ** ns * ** ns ns
Notes: Each value is the mean ± SE of three replicates. Different letters followed values designate significant differences (Duncan’s test, P ≤ 0.05). *, ** Significant at P < 0.05 and P < 0.01, respectively. WS − water stress; ns − not significant.
plants lacking mycorrhiza had higher enzyme activity under MWS, compared with those having mycorrhiza, this difference was not statistically significant (Table 6). In general, chickpea plants having mycorrhiza had sig nificantly more APX activity than those without myc orrhiza (Table 6). Activity of APX enzyme in cv. Pirouz was considerably higher than that in ILC482 (Fig. 2). At pod initiation, chickpea plants of both cultivars under SWS showed significantly higher CAT activity compared with the plants under MWS. However, CAT activity for plants under MWS and WWC was statisti cally similar (Fig. 3, Table 2). Under MWS and WWC conditions, there was no significant difference in CAT activity of Pirouz (Desi type) and ILC482 (Kabuli type) between NAM—NAM and AM—AM plants. Under severe drought stress, the Kabuli cultivar lacking AM had higher CAT activity. In other irrigation treat
ments, there was no significant difference among chickpea cultivars having mycorrhiza and those lack ing it (Table 2). In general, NAM plants had signifi cantly higher CAT activity than AM plants (Fig. 4). Drought stress had no significant effect on POD activity (data not shown). The inoculation of chickpea with AM caused an increase in the activity of POD enzyme, as the activity of this enzyme was significantly higher in AM plants than in NAM plants (Fig. 4). Lipid peroxidation (MDA) significantly varied between AM and NAM chickpea plants under all irri gation treatments (Table 5). Under SWS and MWS conditions, plants lacking mycorrhiza (NAM) showed more active lipid peroxidation, but under WWC con ditions chickpea plants inoculated with G. etunicatum had more active lipid peroxidation. Comparisons among different irrigation levels revealed that chick
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a
a
14
APX activity, mmol ascorbate oxidized/(mg protein min)
Proline content, mg/g dry wt
16
12 10
713
b
8 6 4 2
100 80 60 40 b 20 0
0 SWS
MWS Irrigation level
Pirouz
WWC
ILC482 Cultivar
Fig. 1. Proline content in chickpea leaves subjected to dif ferent irrigation treatments. Each value is the mean ± SE of three replicates (Duncan’s test, P ≤ 0.05).
Fig. 2. Ascorbate peroxidase (APX) activity in leaves of two chickpea cultivars (Pirouz and ILC482). Each value is the mean ± SE of three replicates (Duncan’s test, P ≤ 0.05).
pea NAM plants under drought stress (MWS and SWS) had the highest level of lipid peroxidation than AM plants (Table 5). Interaction between chickpea cultivars and irrigation levels (Fig. 5) indicated that under SWS and MWS conditions, cv. Pirouz had more active lipid peroxidation compared with cv. ILC482, whereas two cultivars had no significant difference under wellwatering (WWC). In general, lipid peroxi dation in cv. Pirouz was more active than that in cv. ILC482 (Fig. 5).
in leaves and roots were higher in wellwatered in AM than in wellwatered NAM seedlings. Drought stress increased proline accumulation in chickpea plants (Table 3). Plants can partly protect themselves against mild drought stress by accumulating osmolytes. Pro line is one of the most common compatible osmolytes in drought stressed plants. Similar result was reported by Alexieva et al. [22].
DISCUSSION Inoculation of chickpea cultivars with G. etunica tum increased leaf chlorophyll content under well and limited irrigation conditions. This improvement for cv. Pirouz was more evident under water stress conditions (Table 2). This is possibly due to the fact that, at low soil water potential, mycorrhizal plants can absorb more water than nonmycorrhizal ones, as mentioned by SánchezBlanco et al. [20] who detected the higher chlorophyll content in mycorrhizal plants subjected to drought stress, which could be associated with the higher photosynthesis rate and plant growth. The AM plants had the comparatively higher leaf soluble protein content under well watering. Reduced protein in the leaves of plants colonized by G. etunica tum than noncolonized plants under MWS condition (Table 5) showed that oxidative stress caused by drought was reduced by colonization with AM. In spite of the increased soluble protein content in leaves of AM plants under drought stress conditions, no sig nificance difference was found between AM and NAM plants. Wu et al. [21] also showed that soluble proteins RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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In plants, the metabolism of ROS, such as superox •– ide radical ( O 2 ), hydrogen peroxide (H2O2), and hydroxyl radical (OH•) is kept in dynamic balance. Under water stress conditions, the balance is broken and antioxidant systems are needed to decrease the damage to tissues [23]. The induction of ROSscav enging enzymes, such as CAT, POD, and APX, are the most common mechanisms for detoxifying ROS syn thesized during stress responses. The chickpea plants under severe stress had the highest CAT activity (Table 2, Table 6. Orthogonal comparisons for amounts of polyphe nol oxidase (PPO) and ascorbate peroxidase (APX) activi ties in chickpea leaves in plants with (AM) and without (NAM) mycorrhiza application Mycorrhi za status NAM AM
Antioxidant enzyme activity PPO, APX, mmol ascorbate U/(mg protein min) oxidized/(mg protein min) 7.008 ± 0.585b 18.368 ± 2.432a
43.760 ± 13.031b 85.910 ± 18.011a
Note: Each value is the mean ± SE of three replicates (Duncan’s test, P ≤ 0.05). No. 6
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a
30 25 20 15 10 5
b 1 bc
2 c
bc c
0 WWC
MWS Irrigation level
SWS
Enzyme activity, U/(mg protein min)
SOHRABI et al. CAT activity, U/(mg protein min)
714
1 a
16 14 12 10 8 6 4 2 0
b
a
2 b
NAM AM Mycorrhiza status
Fig. 4. Orthogonal comparisons for amounts of catalase (1) and peroxidase (2) activities in chickpea leaves in plants with (AM) and without mycorrhiza (NAM) application. Each value is the mean ± SE of three replicates (Duncan’s test, P ≤ 0.05).
Fig. 3). CAT, which is present only in peroxisomes, dismutates H2O2 into water and molecular oxygen [24] and prevents its destructive effects. According to Por cel et al. [8], AM protection of host plants against oxi dative damage was due to the increment in the enzy matic antioxidant activities. The data obtained in the present study suggests that in general, mycorrhizal plants, whether exposed to drought stress or not, showed the lower CAT activity (Table 2). POD (Fig. 4) and PPO (Table 6) activities were sig nificantly higher in AM than in NAM chickpea plants. He et al. [25] stated that POD activity in AM tomato roots was significantly higher than in corresponding NAM plants under saline or saltless conditions. Thus, AM colonization could somewhat enhance the activities of antioxidant enzymes (e.g., POD, PPO, and APX) in chickpeas leaves. This might indicate that the plants inoculated with AM fungi also developed mechanisms to avoid oxidative damage produced under drought stress conditions. Thus, AM plants may have important ecological implications for adaptation to adverse environmental conditions. AM symbiosis (G. etunicatum) notably increased APX activity in chickpea leaves under SWS condition (Table 6). Wu et al. [21] also detected that AM infec tion markedly increased the APX activity of Poncirus trifoliate leaves under drought stress. APX can scav enge H2O2 efficiently in chloroplasts [18]. Further more, He et al. [25] showed that APX activity in tomato roots inoculated with AM was significantly higher than in corresponding NAM plants in saline or saltless conditions. The alteration of membrane phospholipids through lipid peroxidation is considered to be one of the primary key events in oxidative damages. The determination of MDA in a variety of abiotic stress conditions has also been used frequently to assess the
extent of tissue damage caused by lipid peroxidation [26]. The data of the present study revealed that lipid peroxidation in chickpea plants significantly increased under drought stress (Fig. 6). The level of lipid perox idation under severe stress conditions was lower in AM plants (Table 5). Porcel et al. [8] reported that AM soy bean had lower oxidative damage to lipids under drought stress, which is similar to our results. This may be due to the enhanced antioxidative enzyme activi ties induced by AM invasion. He et al. [25] also reported that MDA content in AM seedlings was sig nificantly lower than that in NAM plants under salin ity stress. Since membrane lipid peroxidation was lower in AM plants compared with NAM plants under drought stress, an increas in the APX, POD, and PPO activities in AM plants may be an important mechanism to
MDA content, nmol/g fr wt
Fig. 3. Catalase (CAT) activity in leaves of two chickpea cultivars (Pirouz and ILC482) subjected to different irriga tion treatments. (1) Pirouz, (2) ILC482. Each value is the mean ± SE of three replicates (Duncan’s test, P ≤ 0.05).
6
a a
5 4 3
1 b
b
2 b
b
2 1 0 WWC
MWS Irrigation level
SWS
Fig. 5. MDA content in leaves of two chickpea cultivars (Pirouz and ILC482) subjected to different irrigation treat ments. (1) Pirouz, (2) ILC482. Each value is the mean SE of three replicates (Duncan’s test, P ≤ 0.05).
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4.5
a
a 3.
4.0 3.5
4.
b
3.0
5.
2.5 2.0 6.
1.5 1.0 0.5 0 SWS
MWS Irrigation level
7.
WWC
Fig. 6. MDA content in chickpea leaves subjected to differ ent irrigation treatments. Each value is the mean ± SE of three replicates (Duncan’s test, P ≤ 0.05).
improve drought resistance of AM plants. However, the exact mechanisms involved are still unclear and further experiments in this direction are needed for better understanding the actual function of AM in the changes of ROS metabolism and antioxidant produc tions. Under drought stress, lipid peroxidation in cv. ILC482 was less active than that in cv. Pirouz (Fig. 5). This could be attributed to the better developed pri mary droughtavoidance mechanisms, such as the active water transfer from AM fungi to the host or increased water uptake related to mycorrhizal changes in root morphology of Kabuli type cultivar.
8.
9.
10.
11. 12.
CONCLUSIONS Inoculation of chickpea plants with G. etunicatum increases leaf chlorophyll content and POD and PPO antioxidant enzyme activities. This can also enhance APX activity in plants under severe water stress. Inoc ulation of plants with mycorrhizal fungi results in the lower MDA content under drought stresses (MWS and SWS). A decrease in the MDA content in chickpea plants with AM under stress can be attributed to the increasing activities of the antioxidant enzymes. Thus, AM symbiosis may be an important mechanism to improve drought resistance of chickpea plants.
13.
14. 15.
16.
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