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Agronomic effectiveness of mycorrhizal Cicer arietinum L. plants on mechanism of proline in imparting protection, against NaCl stress.

INTRODUCTION

Plants are exposed to a variety of abiotic stresses such as drought, low and high temperatures, salinity, water logging, radiation, flooding, heavy metals and high light. These stresses are responsible for the greatest agricultural losses worldwide [48,5]. High salinity, most commonly mediated by NaCl, is one of the major abiotic stresses globally. Increased salinization of arable land is expected to have devastating effect, resulting in 30 per cent land loss within the next 25 y. and up to 50 per cent by the middle of the 21st century [72,17]. Legume roots are exposed to arrange of soil microorganisms with which they form a variety of interactions such as Rhizobum and arbuscular mycorrhizal (AM) symbioses. Arbuscular mycorrhiza (AM) fungi are known to occur naturally in most saline soils. AM association increases host plant resistance/tolerance against abiotic stresses, including salinity. Thus, profitable use of AM requires selection of a suitable combination of host plant and fungal partner [7,21]. Most legumes have the ability to establish mutualistic symbiotic relationships with soil N-fixing bacteria (collectively known as rhizobia) and arbuscular mycorrhizal fungi. The free amino acid proline is one of the most widely distributed compatible solutes that accumulate in plants and bacteria during adverse environmental conditions. Proline, an amino acid, which is elevated in response to diverse types of abiotic stresses [68,37,27] is one such molecule that has several roles such as turgor generation, storage of carbon and nitrogen, as partial antioxidant (Smirnoff and Cumbes, 1989), molecular chaperone stabilizing the structure of proteins, maintenance of cytosolic pH, balance of redox status and as part of stress signal [43] influencing adaptive responses (Verbruggen and Hermans, 2008). Pyrroline-5- carboxylate synthetase (P-5-CS) is a bifunctional enzyme (EC 2.7.2.11/1.2.41) that catalyzes the first two steps of the glutamate pathway in proline biosynthesis in plants. Activity of P-5-CS in the chloroplasts can recycle [NADP.sup.+], the last acceptor of the photosynthetic electron transfer chain, which may reduce ROS production at the photosystem I [65]. Another possible route for generating glutamate in the mitochondria is glutamate dehydrogenase (GDH; EC 1.4.1.3), which can either directly produce glutamate from ammonium and a-ketoglutarate or deaminate glutamate to produce a-ketoglutarate. Although the role of GDH is not clear, there is evidence that it can be important in supplying glutamate for proline synthesis [23,69,9]. Oxidative degradation of proline to glutamate is carried out in the mitochondria by sequential actions of proline dehydrogenase (ProDH) and P5C-dehydrogenase (P5C-DH) [65]. Transcriptome analysis using real time- reverse transcrip-tase indicated that mycorrhiza treatment resulted in an increase in the gene expres-sion of P-5-CS on the transcription level [3]. Chickpea is the largest produced food legume in South Asia and the third largest produced food legume globally, after common bean (Phaseolus vulgaris L.) and field pea (Pisum sativum L.). Chickpea forms effective association with both rhizobia as well as AM [66] because of its prolific and deep rooting characteristics [28,13]. Hence, in the present study, we aimed at exploring the role of F. mosseae in NaCl-induced changes associated with proline metabolism, [Na.sup.+] ion uptake, and plant growth in Cicer arietinum L. (chickpea) genotypes as a mode.

MATERIALS AND METHODS

Experimental material consisted of two genotypes of chickpea, salt-tolerant (PBG 5) and salt-sensitive (CSG 9505), procured from the Department of Vegetable Crops, College of Agriculture, Punjab Agricultural University (PAU), Ludhiana, India, and from the Central Soil Salinity Research Institute (CSSRI), Karnal, India, respectively. Mycorrhizal inoculum of F. mosseae (UTMU 128 WM1/1) was obtained from The Energy and Resource Institute (TERI), New Delhi, India. The inoculum was bulked in an open-pot soil culture using Zea mays L., Cajanuscajan (L.) Millsp., Sorghum bicolor L., and Coriandrum sativum L. Experiments were conducted in Panjab University, Chandigarh (30.5[degrees] N, 76.5[degrees] E; elevation = 305-366 m), from November 2012 to March 2013 (minimum temperature, 11-14 [degrees]C; maximum temperature28-32 [degrees]C; morning relative humidity, 28 %; and afternoon relative humidity, 86 %). All results were statistically analyzed by analysis of (ANOVA) using SPSS 18.0 for Windows (SPSS, Inc., Chicago, IL, USA). Duncan's multiple- range test was performed at p < 0.05 on each of the significant variables measured.

Experimental Design:

A mixture of sand and loam in ratio of 1:1 by volume was obtained from nearby agricultural fields. It was autoclaved (121[degrees]C, 1 h twice at 48-h interval) to eliminate existing AM fungi propagules. Seeds were inoculated with fungal inocula of F. mosseae, placed in a pot at 1.5 cm depth prior to sowing. Fifty grams of soil-based inoculum (containing 60-70AM propagules per 10 g soil) along with chopped AM- colonized roots of trap plants were added to each pot. Non-AM treatments received the same weight of autoclaved inoculum (to obtain the same soil texture) together with a 10-mL aliquotof an inoculum filtrate. Seeds were surface-sterilized with 10 % hydrogen peroxide (v/v) solution for a few minutes and then rinsed by soaking in sterile distilled water. After 2 weeks of seedling establishment, NaCl was applied as a water solution of NaCl (0, 4, 6, and 8 dS [m.sup.-1] ECe corresponding to 0, 40, 60, and 80 mM NaCl, respectively) (Richards 1954), with and without AM inoculations.

Methodology:

Mycorrhizal Dependency (MD):

An index of mycorrhizal dependency (MD) was determined by expressing the dry weights of the plants concerned as a percentage of the dry weight of the control plants [18].

Pyrroline-5-carboxylate Synthetase (P-5-CS):

Pyrroline-5-carboxylate synthetase activity was assayed as described by Garcia- Rios et al. [24]. The reaction mixture (3 mL) contained 100 mM Tris-HCl buffer (pH 7.2), 25 mM MgCl2, 75 mM sodium glutamate, 5 mM ATP, and 0.2 mL of enzyme extract. The reaction was initiated by the addition of 0.4 mM NADPH. The activity was measured as the rate of consumption of NADPH monitored by the decrease in absorbance at 340 nm.

Glutamate dehydrogenase (GDH):

Glutamate dehydrogenase (GDH) was assayed by the method of Pahlich and Joy [51]. Enzyme extract was added to 2 ml of tris-HCl buffer (pH 8.0). In the sequence, 0.2 ml a- ketoglutaric acid and 0.2 ml of ammonium sulfate were added. The reaction was started by adding 0.5 ml of NADH. The tubes were incubated at 37[degrees]C for 30 min. The activity was measured as the rate of consumption of NADH monitored by decrease in absorbance at 340 nm, and was expressed as [micro]kat mg-1 protein.

Proline Dehydrogenase (ProDH):

Proline Dehydrogenase (ProDH) Proline dehydrogenase activity was assayed according to the method of Reno and Splittstoesser [58]. The reaction mixture contained 100 mM Na2CO3- NaHCO3 (pH 10.3), 20 mM Lproline, and 0.5 mL enzyme extract in a final volume of 3 mL. The reaction was initiated by the addition of 10 mM NAD. The increase in absorbance at 340 nm was measured at 32 [degrees]C.

Proline content:

The proline content was estimated using the acid ninhydrin method [10]. The leaf tissue was homogenized with 6 ml of 3% (w/v) sulfosalicylic acid aqueous solution and the homogenate was filtered through Whatman No. 1 filter paper. Two ml of the filtered extract was taken for the analysis to which 2 ml acid ninhydrin and 2 ml of glacial acetic acid were added. The reaction mixture was incubated in a boiling water bath for 1 h and the reaction was finished in an ice bath. Four ml of toluene was added to the reaction mixture and the organic phase was extracted, in which a toluene soluble reddish chromophore was obtained, which was read at 520 nm using toluene as blank by UV-visible spectrophotometer.

Chlorophyll:

Extraction of chlorophyll was carried out in dimethyl sulphoxide (DMSO) using leaf discs, following the method of Hiscox and Israelstam [34]. The absorbance of chlorophyll in DMSO was measured at two wavelengths, 645 and 663 nm, in a spectrophotometer against DMSO.

Relative membrane permeability:

The electrolyte leakage was determined to assess membrane permeability as described by Zwiazek and Blake (1991). The plant samples were washed with distilled or de-ionized water to remove surface contamination. 2.5 g of fresh plant material was placed in 25 ml of deionized water for 10 min and subsequently, the electrolytic conductivity of the bathing solution was measured with a conductivity meter. The plant material was then heated to boiling. The bathing solution was cooled to room temperature, and electrolytic conductivity was measured again. The electrolyte leakage was calculated according to the following equation:

Electrolyt e Leakage (%) = [Electrolytic conductivity of solution before heating / Electrolyt ic conductivity of solution after heating] x 100

Thiobaribituric acid-tbars:

Lipid peroxidation is oxidative degradation of lipid-fatty acids by reactive oxygen species (ROS). The level of lipid peroxidation is measured in terms of thiobarbituric acid reactive substances (TBARS) content (Heath and Packer, 1968). Lipid peroxidation products like malondialdehyde, fatty acid- hydro-peroxides react with thiobarbituric acid and form a red colour complex, known asthiobarbituric acid reactive substance, which is taken as the measure of in vivo lipid peroxidation in plant tissue. This red coloured complex absorbs at 532 nm.

Sodium Content:

Sodium ([Na.sup.+]) content was estimated using flame photometer according to method of Chapman and Pratt (1961). First, 10 mL of acid mixture consisting of nitric acid, sulfuric acid, and perchloric acid at a ratio of 9:4:1 was added to 2-5 g of ground samples and kept at 120 [degrees]C overnight. The samples were then maintained at 70 [degrees]C on a hot plate for 30 min; the temperature was increased to 120 [degrees]C for 30 min and then to 250 [degrees]C until only 3 -4 mL of the sample was left. A final volume of 50 mL was maintained using distilled water and left overnight. The next day it was filtered using Whatman No. 1 filter paper. A blank was run without plant samples.

RESULTS AND DISCUSSIONS

NaCl decreased dry matter production of chickpea plants and marked detrimental effects of increased levels of salinity on growth were observed in PBG-5 and CSG-9505. There was severe yellowing of leaves and necrosis of leaf margins, at the highest saline concentration in CSG-9505. Salinity (S), genotype (G), and arbuscular mycorrhizal (AM) inoculations, when considered individually, had significant effects on all the different parameters studied (Table 1). Analysis of combined effects showed that although S x G had significant effects on all the parameters, S x AM was mainly significant with the exception of relative member permeability. The interaction between AM and G and the interaction of all three factors (S x AM x G) with each other were significant, except for shoot dry weight and relative member permeability between AM and G, then chlorophyll content (chlorophyll a and b), shoot dry weight and relative member permeability in S x AM x G (Table 1).

Dry weight of Shoots (SDW):

Both genotypes showed obvious differences in SDW in response to salt stress, with PBG 5 being more tolerant than CSG 9505. There was severe yellowing, necrotic spots on leaf tips and necrosis of leaf margins and drying of growing tips. Shoot biomass depressed significantly by increasing salinity level in both species. The adverse effects of salinity were more apparent at the higher salinity level of 8 dS [m.sup.-1] than when the plants were subjected to the lower saline dosage of 4 dS [m.sup.-1]. A decline of 52.55% under the salt dosage of 6 dS [m.sup.-1] and 73.38% under higher salt dosage (8 dS [m.sup.-1]) was observed in CSG-9505 relative to the unstressed control at 80 DAS. The dry weights of roots declined by 32.96% under 6 dS [m.sup.-1] and Rising root zone salinity from 6 dS[m.sup.-1] to 8 dS[m.sup.-1] caused decrement of 50.27% in PBG-5 over controls at 80 DAS. Inoculations with F. mosseae improved root and shoot dry weights under saline conditions (Table 2, 3). The growth response to AM fungal colonization was more effective in improving shoot growth, and particularly under salt stress (Table 2, 3). Total amelioration of the negative effects of salt stress was absorbed at 4 dS [m.sup.-1], and shoot biomass was greater than untreated controls in PBG-5. This was substantiated by the higher plant biomass in the salt-tolerant genotype PBG-5 than in the salt-sensitive genotype CSG-9505. Salinity stress led to a significant decline in plant shoot biomass in two chickpea genotypes. Our results coincided with those of Tejera et al. (2006) and of Garg and singla (2004) who reported depressive effects of salt dry matter accumulation at the genotypic level in Cicer arietinum L.

Chlorophyll (chl) content:

PBG 5 always exhibited better photosynthesizing capacity due to comparatively higher content of green porphyrin-ring pigment than CSG 9505. A reduction of 39.72 and 32.25 per cent was observed in Chl a and b content respectively in PBG-G under 8 dS[m.sup.-1]. On the contrary, CSG 9505 plants lost the photosynthetic pigment to a greater extent and a declension of as much as 61.29 and 54.12 per cent was noticed in Chl a and b contents respectively under 6 dS[m.sup.-1]. Higher concentrations of NaCl (8 dS[m.sup.-1]) was more deleterious to Chl a degeneration, thus accounting for lesser Chl a/b ratio, than their lower concentrations (i.e. 4 and 6 dS[m.sup.-1]). Mycorrhizal plants had greener leaves than uncolonized plants even under stresses. AM plants of PBG 5 under control conditions had 7.93 and 12.51 per cent higher content of green pigments, Chl a and b content respectively than non-AM unstressed plants. AM symbiosis could enhance the Chl a and b concentration by 4.11 and 10.75 per cent respectively in leaves of CSG 9505 over unstressed non- AM counterparts. Application of mycorrhizal inoculum resurrected the photosynthetic ability of PBG 5 under 6 and 8 dS[m.sup.-1] by causing enhancement of 22.02 and 27.39 per cent (6 dS[m.sup.-1]) and 27.28 and 34.60 per cent (8 dS[m.sup.-1]) in photosynthetic pigments, Chl a and b respectively over the corresponding non-AM stressed plants. meliorism of 14.76 and 22.88 per cent (6 dS[m.sup.-1]) and 21.38 and 29.20 per cent (8 dS[m.sup.-1]) were recorded in Chl a and b content respectively in leaves relative to non-mycorrhizal stressed controls under 6 dS[m.sup.-1] and 8 dS[m.sup.-1] respectively. AM inoculations also decreased the unfavourable effects of NaCl stress on Chl a/b ratio. Thus, AM colonized plants had comparatively higher values of Chl ratio as compared to non-AM plants. The photosynthetic capacity of plants grown under saline conditions is depressed depending on type of salinity, duration of treatment, species and plant age [62,49,63,15]. The reduction in photosynthesis under salinity can also be attributed to a decrease in chl. content [36,47]. Salinity significantly reduces the chl. content and the degree of reduction in chl. depends on salt tolerance of plant species and salt concentration. Salt stress also affects photosynthetic components [53]. Salinity significantly reduces the total chl. content and the degree of reduction in total chl. depends on salt tolerance of plant species and salt concentration. Sabra et al. [61] observed reduced chl a, chl b and carotenoid contents in Echinacea purpurea and E. angustifolia, and this reduction was correlated with shoot sodium ([Na.sup.+]) content rather than chloride (Cl-), suggesting that [Na.sup.+] was the major ion causing pigment reduction. Reduction in growth and photosynthesis may be partly due to lower potential in the cells which, in turn, causes stomatal closure, and reduce transpiration. Thus, limited CO2 availability can alter leaf carbohydrate content and source-to-sink translocation pattern [52,35,6,50]. The reduction of chlorophyll a and b amounts with NaCl application maybe due to increased activity of destructive enzymes called chlorophyllase [57,8,55,56]. Removal of water from the cytoplasm to the extracellular space causes a decrease in the cytosolic and vacuolar volumes [71] and a reduction in the rate of photosynthesis [2,16]. Mycorrhizal colonization also significantly improved chlorophyll content as well as water status in F. mosseae-inoculated PBG 5 plants resulting in higher photosynthesis under saline-amended soils. The rapid process of grain filling was enhanced by fungal endophyte which accounted for higher accumulation of photoassimilates in the relatively greener and expanded leaves, thereby, greater biomass and yield in PBG 5 [12,1,30].

Mycorrhizal symbiosis:

Mycorrhizal dependency can be defined as the degree to which a plant is dependent on mycorrhizal condition to produce its maximum growth or yield, at a given level of soil fertility. The results of the MD calculation showed that PBG 5 had 31.46 per cent dependence under 4 dS[m.sup.- 1], 33.40 per cent at 6 dS[m.sup.-1] which further increased to 37.73 per cent under 8 dS[m.sup.-1] at 80 DAS. On the contrary, MD of 34.18 per cent under 4 dS[m.sup.-1], 37.00 per cent at 6 dS[m.sup.-1] and 40.34 per cent under 8 dS[m.sup.-1] was recorded in CSG 9505 at 80 DAS. Thus, these results evidenced the high mycorrhizal dependence of chickpea genotypes to reach optimum development particularly under stress conditions as well as the efficiency of F. mosseae in protection offered against the detrimental effects of NaCl stress. The results provided evidence of higher mycorrhizal dependence of CSG 9505 to reach optimum development under stress conditions as compared to PBG 5. Symbiotic development between mycorrhizal fungi and chickpea plants seemed to be strengthened in stressed environment after the establishment of association indicating ecological importance of AM for plant survival and growth of plants under stress [41,45,1].

Proline Metabolism:

The increase in proline had a direct correlation with increased [Na.sup.+] content in leaves of both genotypes (rP: PBG-5 = 0.795, CsG-9505 = 0.752) (Fig. 1). The quantitative analysis of proline revealed significantly higher levels of this amino acid at 8 dS [m.sup.-1] than at 6 and 4 dS [m.sup.-1] in leaves. Salinity stress led to a higher increase in the activity of enzyme of the proline biosynthetic pathway in leaves with P-5-CS and GDH (Fig. 2, 3) activity. The induction of P-5-CS and GDH was correlated with the augmentation of salt concentration of the irrigation solution in leaves [P-5-CS ([r.sub.P]): PBG-5 = 0.854, CSG-9505 = 0.781; GDH ([r.sub.P]): PBG-5 = 0.813, CSG-9505 = 0.728]. PBG-5 accumulated large amounts of P-5-CS in leaves when compared with CSG-9505, indicating its higher potential for osmotic adjustment. On the other hand, salinity induced a remarkable reduction in proline dehydrogenase (Fig.4), suggesting that accumulation of P-5-CS was the result of concomitant inactivation of proline dehydrogenase, thus negatively correlated with sodium content in leaves ([r.sub.P]: PBG-5 = 0.759, CSG-9505 = 0.724). AM colonization with F. mosseae had a positive influence on the activity of P-5-CS, thus, AMinoculated plants showed higher proline synthesis (P-5-CS, GDH) at all salinity levels, with a further decline in PDH activity. Association of chickpea roots with F. mosseae resulted in a conspicuous increase in the proline pool under salt stress as shown by the positive correlation of proline with plant MD [leaves (rP): PBG-5 = 0.937, CSG-9505 = 0.917]. The S x AM * G interaction for proline content, P-5-CS as well as GDH (Table 1) confirmed a significant and a positive role of AM in modulating the tolerance response to NaCl in chickpea genotypes. The decrease in soil water potential under salt stress might have led to an alteration of the plant water status, which might have caused stomata closure, photosynthesis reduction, and thus growth inhibition [4]. The change in proline biosynthesis with higher activity of biosynthetic enzyme (P-5- CS) with a concomitant decline in ProDH activity, in PBG 5 could be correlated with its capacity to tolerate and adapt to salinity condition [72]. Transcriptome analysis using real-time reverse transcriptase by Abo-Doma et al. [3] indicated that mycorrhiza treatment resulted in an increase in the gene expression of P-5-CS on the transcription level. Thus, PBG 5 plants with better stress tolerance had efficient osmoregulation mechanisms to avoid fluctuations in their cytosol in order to maintain growth under stressful environment (NaCl).

Relative member permeability:

An alternation in leaf plasma membrane integrity was induced on addition of 4 dS[m.sup.-1] due to 10.3 per cent increase in membrane permeability over controls in PBG 5. Salinity stress of 6 and 8 dS[m.sup.-1] accelerated membrane injury causing an upsurge of 18.49 and 30.91 per cent in membrane leak over unstressed control plants in PBG 5. Application of 4 dS[m.sup.-1] caused a higher accretion of 18.50 per cent in loss of selectivity relative to unstressed controls in CSG 9505. Severe impairment of membrane functioning and selectivity with as much as 38.69 and 53.26 per cent increase in cellular membrane leakage in CSG 9505 was recorded under 6 and 8 dS[m.sup.-1] respectively. Augmentation of 5.28 per cent in the maintenance of physical state of membranes was observed under 4 dS[m.sup.-1] in AM plants of PBG 5 than the corresponding non- AM stressed plants. AM colonization had positive impact on selective functioning of physical barrier (membrane) and AM plants had 16.38 and 18.11 per cent higher membrane integrity under 6 and 8 dS[m.sup.-1] in PBG 5 comparative to stressed nonAM controls. Increment percentage of 10.09, 13.71 and 16.49 was recorded in reducing membrane flux of electrolytes under 4 dS[m.sup.-1], 6 dS[m.sup.-1] and 8 dS[m.sup.-1] respectively in CSG 9505 relative to non-AM stressed counterparts. PBG 5 however, recorded less loss of membrane functionality than CSG 9505 under NaCl stress, which might be due to its genetic potential to accommodate the impact of soil stress. There are reports that salinity enhances electrolyte leakage and peroxidation of lipid membrane, disrupting its permeability in a tissue-specific [31,39] and genotypic manner [60,59,73]. The intensity of membrane damage was lower in AM inoculated plants as compared to non-AM plants. Less damage in membrane system of AM plants was reflected in lower electrolyte leakage, which in turn was due to lower peroxidation of membranes in AM plants under salt stress. Until now, salt stress tolerance studies have recommended that mycorrhizal plants grow better due to improved mineral nutrition and physiological processes like photosynthesis or water use efficiency [44,19,20].

Sodium ([na.sup.+]) content:

The ion-specific phase of plant response to salinity started when [Na.sup.+] accumulated to toxic concentrations in the plant tissues which was linked to loss of membrane selectivity and functionality as the stress intensified. The concentration of [Na.sup.+] significantly increased in the leaves of chickpea plants in the presence of NaCl stress (Table 2, 3). CSG-9505 showed higher accumulation of [Na.sup.+] when compared to PBG-5 and thus, higher sensitivity to [Na.sup.+] ions. It was seen that most of the [Na.sup.+] was held up in the roots and much less reached the leaves in both genotypes. Mycorrhization with F. mosseae counteracted such harmful accumulation of [Na.sup.+] in two genotypes of chickpea. A gradient accretion was observed in [Na.sup.+] content when salinity was raised from 4 to 6 and 8 dS [m.sup.-1], with higher increase at 8 dS [m.sup.-1]. At the high salinity level (8 dS [m.sup.-1]), tissues showed the inability to control [Na.sup.+] uptake and transport where the ionic stress dominated. There was a positive correlation ([r.sub.P]: PBG-5 = 0.956, CSG-9505 = 0.989) between sodium content and mycorrhizal dependency. Thus, mycorrhizal plants had significantly lower [Na.sup.+] content than nonmycorrhizal plants under stressed conditions. PBG-5 was able to recover from ionic stress and had less [Na.sup.+] content upon AM colonization than did CSG9505. Higher [Na.sup.+] concentration was found in the sensitive genotype (CSG 9505) when compared with the tolerant one (PBG 5) suggesting a positive correlation between salt tolerance and the control of [Na.sup.+] absorption. The way to adapt to salinity stress is to keep cytosolic [Na.sup.+] level low at the cellular level and to keep shoot [Na.sup.+] concentrations low at the whole plant level. genotypic differences observed in the present study in plant growth response to NaCl stress depended on the intrinsic differences in rates of toxic ion uptake, transport and distribution within the plant [40,11,42,22,29]. Differential mycorrhizal responsiveness of genotypes could be the result of plant or fungus related mechanisms. Enhancement of growth in mycorrhizal plants under saline conditions has been related partly to mycorrhizal-mediated enhancement of host plant P nutrition and other nutrients with low mobility, such as Fe, Cu, and Zn [45,39] and decreased uptake of [Na.sup.+] [32, 26].

Thiobaribituric acid (tbars):

Lipids are among the most prominent constituents of cell membrane which play a fundamental role in cell permeability. Lipid peroxidation causes degradation and impairment of structural component. This leads to change in selective permeability of bio-membranes and thereby membrane leakage and change in activities of enzymes bound to membrane. The accumulation of free radicals in stressed plants cause oxidation of polyunsaturated fatty acids (PUFA) in the plasma membrane resulting in the formation of thiobaribituric acid (TBARS). Elevated levels of TBARS are thus a measure of lipid peroxidation and membrane damage. The control of ROS generation and scavenging was adversely affected in salt- sensitive genotype (CSG 9505) which showed higher and lower accumulation of TBARS, than salt-tolerant genotype (PBG 5) with increasing NaCl concentrations. TBARS produced as a result of NaCl induced oxidative stress was a highly damaging, especially at higher NaCl (8 dS[m.sup.-1]) concentration as compared to 6 and then 4 dS[m.sup.-1] in leaves (Table 2, 3). In 6 and 8 dS[m.sup.-1] salinity induced 36.73 per cent and 61.22 rise in TBARS-reactive substances over the unstressed control plants in leaves respectively in PBG 5. The composition and organization of lipids inside the bilayer was disrupted to greater extent in CSG 9505 under NaCl which induced oxidative generation of by-products of ROS metabolism. In CSG 9505, 6 dS[m.sup.-1] and higher saline dosage (8 dS[m.sup.- 1]) resulted in 46.25 per cent and 79.13 increases in oxygenated products of lipid degradation (TBARS) as a result of higher ROS accumulation over the unstressed controls in leaves respectively. The association of plants with mycorrhizal fungi modified the chickpea responses to NaCl induced oxidative stress, thus increasing tolerance in stressed soils. ROS induced peroxidation of membrane lipids was considerably lowered by mycorrhizal colonization when compared with uncolonized chickpea plants, especially in PBG 5. The damage to cellular membrane, implicated in TBARS content, was reduced in mycorrhizal plants of PBG 5 under 4 and 6 dS[m.sup.-1] which experienced 18.51 per cent and 22.22 lesser peroxidation of phospholipids in leaves respectively over the corresponding non-AM stressed plants. The occurrence of activated forms of oxygen (H2O2) and subsequent symptoms of oxidative injury were decreased in colonized plants of PBG 5 due to lower production of malondialdehyde even under 8 dS[m.sup.-1]. Hence leaves had 29.53 per cent lower membrane peroxidation over non-mycorrhizal plants at 8dS[m.sup.-1]. The membrane destabilization attributed to enhanced oxidation of phospholipid bilayer was partially counterbalanced by AM fungi, F. mosseae in CSG 9505. The efficiency of mycorrhizal stressed endosymbiont reduced the degeneration of membrane lipids into peroxides by 14.14 per cent at 4 dS[m.sup.-1], 19.24 per cent at 6 dS[m.sup.-1] and 26.37 per cent at 8 dS[m.sup.-1] in leaves respectively over the non-AM salt-stressed plants in CSG 9505. The present results showed a increase in electrolyte leakage (higher relative membrane permeability), as well as thiobaribituric acid in stressed plant and [Na.sup.+] sequestered in both genotypes. PBG 5 however, recorded less loss of membrane functionality and TBARS than CSG 9505 under NaCl stress, which might be due to its genetic potential to accommodate the impact of soil stress. There are reports that salinity enhances electrolyte leakage and peroxidation of lipid membrane, disrupting its permeability or induce oxidative stress in a tissue-specific [31,38,39] and genotypic manner [60,59,73]. The intensity of membrane damage was lower in AM inoculated plants as compared to non-AM plants. Less damage in membrane system of AM plants was reflected in lower electrolyte leakage, which in turn was due to lower peroxidation of membranes in AM plants under salt stress [21].

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Conclusion:

PBG-5 was more salt tolerant and had greater stress protection through increased proline synthesis which led to reduced sodium ion uptake, membrane lipid peroxidation and higher biomass when compared with CSG9505 under stressed conditions. Mycorrhizal colonization under NaCl treatment induced higher increase in the level of P-5-CS and GDH in PBG 5 which was due to enhanced decline of PDH activity in response to salinity. The induction of proline metabolism under salt stress and its manipulation through AMF symbiosis suggests its promising role in imparting salt tolerance in chickpea plants.

ARTICLE INFO

Article history:

Received 12 October 2014

Received in revised form 26 December 2014

Accepted 1 January 2015

Available online 20 February 2015

ACKNOWLEDGMENT

Thanks are due to Prof. Neera Garg and Dr. Priyanka Singla (Department of Botany, Panjab University, Chandigarh, India) for guidance and support in undertaking the present research.

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Navid Baher

Assistant Professor, Department of Agriculture, Payam Noor University, Tehran, Iran

Corresponding Author: Navid Baher, Assistant Professor, Department of Agriculture, Payam Noor University, Tehran, Iran

E-mail: nvdbaher@gmail.com
Table 1: Result of three-way ANOVA test for independent variables,
including salinity treatment (S), arbuscular mycorrhizal (AM)
inoculations and genotype (G) and interactions among them.

Table 1. Result of three-way ANOVA test for independent variables,
including salinity treatment (S), arbuscular mycorrhizal (AM)
inoculations, and genotype (G) and interactions among them.

SxAMxG    AMxG   SxG   SxAM    G

ns         ns     *     *      *
ns         ns     *     ns     *
ns         *      *     *      *
ns         *      *     *      *
ns         *      *     *      *
*          *      *     *      *
*          *      *     *      *
*          *      *     *      *
*          *      *     *      *
*          *      *     *      *
*          *      *     *      *
ns         ns     *     *      *

SxAMxG    AM     S    Parameter

ns         *     *    SDW
ns         *     *    Relative member permeability
ns         *     *    Chl a
ns         *     *    Chl b
ns         *     *    Chl a/b
*          *     *    MD
*          *     *    [Na.sup.+] content
*          *     *    P-5-CS activity
*          *     *    GfH activity
*          *     *    Proline content
*          *     *    ProfH activity
ns         *     *    TBARS

ns no significant differences.

* Significant differences at 95%

Table 2: Effect of Arbuscular mycorrhizal (AM) inoculations on
Relative member permeability (%), Shoot dry weight
(SDW, [g.sup.-1] plant), Chlorophyll a,b and Chlorophyll a/b (Chi, mg
[g.sup.-1] f.wt.), Mycorrhizal dependency (MD, %>), Thiobaibitaric acid
(TBARS, mbol [g.sup.-1] f.wt.) and Sodium content (mg [g.sup.-1] DW) in
leaves of chickpea (PBG 5) under NaCl stress.

Parameter              Control                     C+AM

Relative       15:14 (de) [+ or -] 0:58   13:88 (e) [+ or -] 0:37
member
permeability

SDW            1:79 (bc) [+ or -] 0:05    2:38 (a) [+ or -] 0:05

Chla           2:26 (ab) [+ or -] 0:03    2:44 (a) [+ or -] 0:02

Chlb           1:51 (ab) [+ or -] 0:09    1:69 (a) [+ or -] 0:02

Chla / Chlb     1:50 (a) [+ or -] 0:04    1:44 (c) [+ or -] 0:02

MD                        -               28:26 (c) [+ or -] 0:47

Na             2:14 (de) [+ or -] 0:05    1:82 (e)  [+ or -] 0:04

TBARS          0:44 (cd) [+ or -] 0:01    0:380 (d) [+ or -] 0:01

Parameter           4 dS [m.sup.-1]          4 dS [m.sup.-1]+AM

Relative       16:70 (abc) [+ or -] 0:59    14:34e [+ or -] 0:25
member
permeability

SDW             1:46 (cd) [+ or -] 0:10    2:03 (b) [+ or -] 0:18

Chla            2:04 (c) [+ or -] 0:01     2:34 (b) [+ or -] 0:03

Chlb           1:37 (cd) [+ or -] 0:039    1:65 (a) [+ or -] 0:04

Chla / Chlb     1:48 (ab) [+ or -] 0:01    1:41 (cd) [+ or -] 0:04

MD                         -               31:46 (b) [+ or -] 0:51

Na              2:74 (c) [+ or -] 0:24     2:31 (d) [+ or -] 0:08

TBARS           0:54 (bc) [+ or -] 0:02    0:44 (cd) [+ or -] 0:05

Parameter          6 dS [m.sup.-1]           6 dS[m.sup.-1]+AM

Relative       17:94 (ab) [+ or -] 0:98    15 (de) [+ or -] 0:61
member
permeability

SDW            1:20 (de) [+ or -] :075    1:78 (bc) [+ or -] 0:14

Chla           1:73 (de) [+ or -] 0:07    2:12 (bc) [+ or -] 0:06

Chlb            1:17 (e) [+ or -] 0:03    1:50 (bc) [+ or -] 0:08

Chla / Chlb     1:47 (b) [+ or -] 0:08    1:41 (cd) [+ or -] 0:07

MD                        -               33:40 (a) [+ or -] 0:53

Na              3:51 (b) [+ or -] 0:03    2:75 (c) [+ or -] 0:05

TBARS          0:60l (b) [+ or -] 0:03    0:46 (c) [+ or -] 0:01

Parameter          8 dS [m.sup.-1]         8 dS[m.sup.-1]+ AM

Relative        19:82a [+ or -] 1:64     16:23bCd [+ or -] 1:28
member
permeability

SDW            0:89 (e) [+ or -] 0:17    1:41 (d) [+ or -] 0:10

Chla           1:36 (e) [+ or -] 0:01    1:74 (de) [+ or -] 0:07

Chlb           1:02 (f) [+ or -] 0:026   1:37 (de) [+ or -] 0:04

Chla / Chlb    1:33 (de) [+ or -] 0:02   1:26 (e) [+ or -] 0:08

MD                        -              37:73 (a) [+ or -] 0:36

Na             4:39 (a) [+ or -] 0:21    3:30 (b) [+ or -] 0:01

TBARS          0:71 (a) [+ or -] 0:03    0:50 (c) [+ or -] 0:01

Table 3: Effect of Arbuscular mycorrhizal (AM) inoculations on
Relative member permeability (%), Shoot dry weight (SDW, [g.sup.-1]
plant), Chlorophyll a, b and Chlorophy II a/b (Clil, mg [g.sup.-1]
f.wt), Mycorahuzal dependency (MD, %), Thiobaribituric add (Tbars,
nmol [g.sup.-1] f.wt) and Sodium content (mg g.sup.-1] DW) in
leaves of chickpea (CSG 3505) under NaCl stress.

Parameter                 Control                    C+AM

Relative member   18.81 (e) [+ or -] 1.62   17.61 (e) [+ or -] 0.95
permeability

SDW               1.76 (b) [+ or -] 0.08    2.36 (a) [+ or -] 0.04

Chla              2.09 (a) [+ or -] 0.05    2.17 (a) [+ or -] 0.02

Chlb              1.44 (a) [+ or -] 0.04    1.59 (a) [+ or -] 0.02

Chla / Chlb       1.44 (a) [+ or -] 0.12    1.36 (b) [+ or -] 0.03

MD                           -              29.88 (d) [+ or -] 0.37

Na                2.28 (e) [+ or -] 0.06    2.13 (e) [+ or -] 0.04

TBARS             0.50 (e) [+ or -] 0.009   0.46 (f) [+ or -] 0.02

Parameter             4 dS [m.sup.-1]         4 dS [m.sup.-1]+AM

Relative member    22.29cd [+ or -] 0.85     20.04de [+ or -] 0.59
permeability

SDW               1.23 (c) [+ or -] 0.05    1.73 (b) [+ or -] 0.15

Chla              1.62 (bc) [+ or -] 0.03   1.76 (b) [+ or -] 0.07

Chlb              1.13 (b) [+ or -] 0.03    1.36 (a) [+ or -] 0.06

Chla / Chlb       1.42 (ab) [+ or -] 0.05   1.32 (c) [+ or -] 0.06

MD                           -              31.46 (b) [+ or -] 0.51

Na                3.37 (cd) [+ or -] 0.10   3.05 (d) [+ or -] 0.21

TBARS             0.62 (cd) [+ or -] 0.06   0.54 (e) [+ or -] 0.04

Parameter             6 dS [m.sup.-1]          6 dS[m.sup.-1]+AM

Relative member   26.08 (a) [+ or -] 0.63   22.51 (bc) [+ or -] 0.27
permeability

SDW               0.83 (d) [+ or -] 0.11     1.30 (c) [+ or -] 0.07

Chla              1.21 (cd) [+ or -] 0.03    1.39 (c) [+ or -] 0.04

Chlb              0.91 (c) [+ or -] 0.02     1.12 (b) [+ or -] 0.03

Chla / Chlb       1.33 (c) [+ or -] 0.05     1.24 (d) [+ or -] 0.11

MD                           -              37.90 (b) [+ or -] 0.32

Na                4.39 (b) [+ or -] 0.23     3.68 (c) [+ or -] 0.08

TBARS             0.74 (ab) [+ or -] 0.02   0.60 (de) [+ or -] 0.02

Parameter             8 dS [m.sup.-1]          8 dS[m.sup.-1]+ AM

Relative member   28.83 (a) [+ or -] 1.58   24.07 (ab) [+ or -] 0.51
permeability

SDW               0.46 (e) [+ or -] 0.10    0.77 (de) [+ or -] 0.20

Chla              0.80 (e) [+ or -] 0.05     0.98 (d) [+ or -] 0.04

Chlb              0.66 (e) [+ or -] 0.02     0.85 (d) [+ or -] 0.03

Chla / Chlb       1.22 (d) [+ or -] 0.03     1.14 (e) [+ or -] 0.05

MD                           -              40.92 (a) [+ or -]  0.57

Na                5.55 (a) [+ or -] 0.14     4.39 (b) [+ or -] 0.14

TBARS             0.91 (a) [+ or -] 0.01    0.67 (bc) [+ or -] 0.01
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