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Journal of Stress Physiology & Biochemistry, Vol. 10 No. 1 2014, pp. 335-346 ISSN 1997-0838 Original Text Copyright © 2014 by Shamshiri and Fattahi
ORIGINAL ARTICLE
Evaluation of Two Biochemical Markers for Salt Stress in Three Pistachio Rootstocks Inoculated with Arbuscular Mycorrhiza (Glomus mosseae)
Shamshiri M.H.* and Fattahi. M.
Hort. Dept., College of Agriculture, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran.
*E-Mail: shamshiri88@gmail.com
Received December 1, 2013
The possible involvement of the methylglyoxal and proline accumulation in leaves and roots of three pistachio rootstocks, cv. Sarakha, Abareqi and Bane baghi, pre-inoculated with arbuscular mycorrhizal fungus (Glomus mosseae) in response to salt stress was studied during a greenhouse experiment in 2013. Six months old pistachio seedlings were exposed to four salinity levels of irrigation water (EC of 0.5 as control, 5, 10 and 15 dS m-1) for 70 days. Methylglyoxal and proline of the roots and leaves were increased by increasing salt stress. The highest concentrations of proline in leaves and roots were recorded in Abareqi rootstock while the lowest concentration was observed in Sarakhs. In general, a negative relationship was obtained between proline and methylglyoxal concentrations in both tissues especially at two highest levels of salinity. A very strong relationship between salinity and measured biochemical markers were found. The level of both biomarkers were reduced in both tissues and in all rootstocks as the effect of mycorrhizal symbiosis. Root colonization percentage was declined as the effect of salinity in Abareqi and Bane baghi and not in Sarakhs.
Key words: Methylglyoxal, Mycorrhiza, Pistachio, Proline, Salt stress
ORIGINAL ARTICLE
Evaluation of Two Biochemical Markers for Salt Stress in Three Pistachio Rootstocks Inoculated with Arbuscular Mycorrhiza (Glomus mosseae)
Shamshiri M.H.* and Fattahi. M.
Hort. Dept., College of Agriculture, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran.
*E-Mail: shamshiri88@gmail.com
Received December 1, 2013
The possible involvement of the methylglyoxal and proline accumulation in leaves and roots of three pistachio rootstocks, cv. Sarakha, Abareqi and Bane baghi, pre-inoculated with arbuscular mycorrhizal fungus (Glomus mosseae) in response to salt stress was studied during a greenhouse experiment in 2013. Six months old pistachio seedlings were exposed to four salinity levels of irrigation water (EC of 0.5 as control, 5, 10 and 15 dS m-1) for 70 days. Methylglyoxal and proline of the roots and leaves were increased by increasing salt stress. The highest concentrations of proline in leaves and roots were recorded in Abareqi rootstock while the lowest concentration was observed in Sarakhs. In general, a negative relationship was obtained between proline and methylglyoxal concentrations in both tissues especially at two highest levels of salinity. A very strong relationship between salinity and measured biochemical markers were found. The level of both biomarkers were reduced in both tissues and in all rootstocks as the effect of mycorrhizal symbiosis. Root colonization percentage was declined as the effect of salinity in Abareqi and Bane baghi and not in Sarakhs.
Key words: Methylglyoxal, Mycorrhiza, Pistachio, Proline, Salt stress
Salinity is an abiotic stress factor that limits plant development (Sengupta and Majumder, 2009). As a result of high salt concentrations, ionic imbalance and hyper-osmotic stress are triggered in plants, which consequently elicit secondary stresses such as oxidative damage (Zhu, 2001).
Salinization of soil is a serious problem and is increasing steadily in many arid and semi-arid parts of Kerman province especially in Rafsanjan region that is thought to be the largest center of pistachio
production in Iran and in the world (Bagheri et al., 2011a). High levels of soil salinity in Rafsanjan region is mainly due to the soluble salts in irrigation water and fertilizers used by pistachio producers annually, low precipitation and high temperature in this region. According to this problem, recognition of tolerant rootstocks of pistachio to salinity has a specific importance and can play a key role in sustainable production.
Arbuscular mycorrhizal fungi (AMF) widely occur
in salty soils (Aliasgharzadeh et al., 2001). Recently, many researchers reported that AMF could enhance the ability of plants to cope with salt stress (Yano-Melo et al., 2003; Rabie, 2005; Jahromi et al., 2008) by improving plant nutrient uptake (Cantrell and Linderman, 2001; Asghari et al., 2005) and ion balance (Zandavalli et al., 2004; Giri et al., 2007), protecting enzyme activity (Rabie and Almadini, 2005; Giri and Mukerji, 2004), and facilitating water uptake (Ruiz-Lozano et al., 1995).
Methylglyoxal (MG), is a potent cytotoxic compound produced spontaneously under physiological conditions from the glycolysis and photosynthesis intermediates, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate (Richard, 1993). Under stress, the rate of glycolysis increases leading to an imbalance in the pathway. Triose phosphates are very unstable metabolites, and removal of the phosphoryl group of these trioses leads to the formation of MG (Richard, 1993). Therefore, spontaneous production of MG is an unavoidable consequence of the glycolysis pathway during different stresses like salinity, drought and cold stresses (Yadav et al., 2005; Singla-Pareek et al., 2006). In plants, MG is detoxified mainly via the glyoxalase system. Besides detoxification of MG, the glyoxalase system could also play a role in oxidative stress tolerance by recycling reduced glutathione that would be trapped non enzymatically by MG to form hemithioacetal, thereby maintaining glutathione homeostasis.
It is well known, that one of the most common responses to water deficit and saline environments is the accumulation of proline (Pro) which acts as a compatible solute, an osmoprotectant, and a protective agent for cytosolic enzymes and cellular organelles (Delauney and Verma, 1993; Bohnert et
al., 1995; Demir, 2004). Additionally, Pro is a nitrogen source available for recovery from stress and for restoration of growth (Trotel et al., 1996). Based on previous researches, accumulation of proline in pistachio under both drought and salinity stresses have been proved (Bagheri et al., 2011b; Kamiab et al., 2012).
Development of salt tolerant plants depend on the basis of physiological, biochemical and molecular markers are recommended and may provide mechanistic understanding the term of tolerance. Hence many metabolic changes are known to occur in plants subjected to salt stress such as proline and methylglyoxal accumulation. The aim of this study was to evaluate proline and methylglyoxal as biochemical markers for salt tolerance of three pistachio rootstocks inoculated with AMF.
MATERIALS AND METHODS
Experimental site
A greenhouse experiment was conducted in 2013 at the Agri-college of Vali-e-Asr university of Rafsanjan (30°23'06" N, 55°55'30" E), at1523m a.s.l.
AM inoculum production
Glomus mosseae (Nicolson & Gerdemann) was propagated in a sterile potted soil cropped with Zea mays L. between June and October 2012. AM fungal inoculum consisted of a mixture of rhizospheric soil from trap cultures containing spores, hyphae and mycorrhizal root fragments.
Soil preparation and seed sowing
The soil used was an autoclaved (121°C for 2 h) sandy loam with the following characteristics: sand 70.2%, silt 14.6%, clay 15.2%, pH 7.2, P 7.4 mg kg-1 soil, K 23.4 mg kg-1 soil, Fe 1.3 ^g.g"1, Zn 0.03 ^g.g"1, Mn 0.13 ^g.g"1 , Cu 0.03 ^g.g"1 and cation exchange
capacity 1.7 dS.m-1. Adequate amount of fertilizers (NH4NO3, K2SO4, MnSO4 and Fe EDDHA) were added to soil, based on soil analysis results.
Seeds of three pistachio rootstocks, P. vera cv. Sarakhs, P. vera cv. Abareqi and Bane baghi (P. eurycarpa x P. mutica) were surface sterilized in 10% sodium hypochlorite for 10 min and then incubated at 25°C on sterile moist cloth for one week. Six germinated seeds were sown in each pot containing 5 kg of autoclaved soil. The number of seedlings per pot was reduced to 4 within 21 days of germination.
Mycorrhizal inoculation
One hundred gram (fresh mass) of inoculum having on average of 90% of infected roots was placed on the soil surface immediately before planting and after placing the germinated seeds on it, seeds were covered with sterilized sand. Control plants received the same amount of an autoclaved inocula. The growth of seedlings continued for 180 days in greenhouse (T: 27±5°C; RH: 30±2%; 12-14 h day light without additional artificial lightening) before the start of salt treatments and during this period, the seedlings were watered every two days up to FC level with distilled water. At the end of this stage, sampled roots showed an average of 85% colonization for Sarakhs, 80% for Abareqi and 90% for Bane baghi.
Salt treatments
Four salt levels including EC of 0.5 (tap water as control), 5, 10 and 15 dSm-1, achieved by adding NaCl in irrigation water. To avoid osmotic shock, salt solution in two higher levels (EC of 10 and 15 dSm-1) was introduced gradually by 2 and 3 steps respectively. Plants were harvested 70 days after the commencement of salt treatments and during this period, they were irrigated every two days 30%
more than predetermined FC level to avoid salt accumulation in the soil.
Root sampling and assessment of arbuscular mycorrhizal colonization
The experiment was terminated by separating shoots from roots days after treatments commencement. Roots were carefully rinsed with running tap water and then the roots of 4 plants in each pot was mixed and cut into 1cm in length segments. Samples for mycorrhizal assessment were prepared according to method of Phillips and Haymann (1970). Roots were boiled for 1 h in 15 % KOH and then washed with tap water. Staining was performed in 0.05 % trypan blue by autoclaving the samples for 15 min. Thereafter, Samples were stored in lacto glycerol [mixture of lactic acid, glycerol, water 1:1:1 (v/v/v)]. Root segments were mounted on glass slides and examined under a compound microscope (CHS, Olympus optical Co., LTD, Japan). Mycorrhizal colonization (abundance of hyphae, vesicles and arbuscules) was estimated using 50 root segments of each sample (Giovannetti and Mosse, 1980).
Sample preparation for MG estimation
Methylglyoxal was estimated basically according to the method of Yadav et al. (2005a) with some modification. About 0.5 g of leaf and root tissue was homogenized in 3 mL of 0.5 M perchloric acid. After incubating for 15 min on ice, the mixture was centrifuged at 4°C for 10 min at 11,000 g. The supernatant was decolorized by adding charcoal (10mgmL-1), kept for 15 min at room temperature, and centrifuged at 11,000 g for 10 min. Before using this supernatant for MG assay, it was neutralized by keeping for 15 min with saturated solution of potassium carbonate at room temperature and centrifuged again at 11,000 g for 10 min. The neutralized supernatant was used for MG
estimation.
Methylglyoxal assay
In a total volume of 1ml, 250 nL of 7.2 mM 1, 2-diaminobenzene, 100 ^L of 5 M perchloric acid, and 650 ^L of the neutralized supernatant were added in that order. The absorbance at 335 nm of the derivatized MG was read after 25 min in a spectrophotometer (PG-instrument, T80, China). The final concentration of MG was calculated from the standard curve and expressed in terms of ^mol.g^FW.
Proline determination
Proline colorimetric determination proceeded based on reaction with ninhydrin (Bates et al., 1973). A 1:1:1 solution of proline, ninhydrin acid and glacial acetic acid was incubated at 100°C for 1 hour. The reaction was arrested in an iced bath and the cromophore was extracted with 4 ml toluene and its absorbance at 520 nm was determined. Proline concentration was calculated with a standard curve and expressed as ^g.g"1 fresh mass. Experimental Design
The experiment was a completely randomized design with three replicates and a factorial combination of two mycorrhiza treatments (with or without mycorrhizal), four salinity levels (S^0.5, S2:5, S3:10 and S4:15 dSm-1) and three rootstocks (Abareqi, Sarakhs and Bane baghi). Data were statistically analyzed by analysis of variance with the MSTATC PROGRAM (Michigan State University, East Lansing, Mich., USA). Probabilities of significance were used to test for significance among treatments and interactions and Duncan's multiple range test at 5% significant level was used to compare means.
RESULTS
All salt levels, AMF treatments and pistachio
rootstocks had significant effect on almost all of measured parameters except root proline by mycorrhiza and leaf MG by pistachio rootstocks. The most significant interactions were recorded for MxS and SxPR (Table 1).
Methylglyoxal levels in leaf
It was found that leaf MG levels increased drastically due to different salt stress treatments in both -M and +M plants but in any salt level, -M plants had higher amount of MG (Fig. 1). In -M plants, Abareqi had the least and Sarakhs had the most leaf MG while the opposite results were obtained with +M plants (Fig. 2).
Methylglyoxal levels in root
In comparison with leaf, accumulation of MG was much lower in root. Regardless of salt treatments and pistachio rootstocks, MG content of -M plants was 16% more against +M plants (Fig. 3). Similar to leaf, MG was increased with increasing in salt stress levels but the results were significant just with S3 and S4. At stress levels of Si and S2, there were no significant differences between rootstocks while in higher levels, Sarakhs and Abareqi showed the highest and lowest MG respectively (Fig. 4). Proline accumulation in leaf
Leaf accumulation of proline in response to salt stress and mycorrhizal symbiosis in different pistachio rootstocks presented in Fig. 5. Despite of Mycorrhizal treatment, increasing concentration of NaCl from EC of 0.5 to 15 dS.m-1, progressively increased proline concentration in leaf tissue of Sarakhs, Bane baghi and Abareqi by about 4.7, 6.3 and 4.8 fold respectively over than control treatment. It is interested to note that cv. Sarahks had significantly lower proline concentration at salt level of S4 in -M and +M treatments compared to the other rootstocks. +M plants of all rootstocks
showed lower proline at salt levels of S3 and S4 in comparison with respected control -M (Fig. 6). Proline accumulation in root
Root proline content in the salt stressed seedlings reached to 88.8 (4.93 folds of control) and 75.8 g-1 FW (3 folds of control) in -M and +M
respectively under stress level of S4 (Fig. 7). At each salinity level, no significant difference was observed between +M and -M plants except at S4 where -M plants had higher amount of proline. No significant difference in root proline was observed between pistachio rootstocks at salinity levels of S1 and S2
but at higher salinity levels, cv. Abareqi had higher proline content (Fig. 8).
Root colonization
None of the pistachio plants in the noninoculated treatments were colonized by G. mosseae. The extent of AM colonization decreased significantly with increase in soil salinity (10, 15 dS m-1) in Abareqi and Bane baghi by 58 and 29% respectively over respected control while root colonization of cv. Sarakhs was not affected by salinity except at S3 level (Fig. 9).
Figure 1. Interaction effects of mycorrhizal symbiosis and salt intensities (EC of 0.5, 5, 10 and 15 dS m-1) on leaf MG levels of pistachio rootstocks. Bars indicate standard error. Colomns with different letters aresignificantly different at P< 0.05.
Figure 2. Effects of mycorrhizal symbiosis on leaf MG levels in three pistachio rootstocks (S: Sarakhs, B: Bane baghi, A: Abareqi) grown 70 days under different salt intensities (EC of 0.5, 5, 10 and 15 dS m-1). Bars indicate standard error. Colomns with different letters aresignificantly different at P< 0.05.
M NM
Figure 3. Effect of mycorrhizal symbiosis on root MG level of pistachio rootstocks. Bars indicate standard error. Colomns with different letters aresignificantly different at P< 0.05.
Figure 4. Effects of different salt intensities (EC of 0.5, 5, 10 and 15 dS m-1) on root MG levels in three pistachio rootstocks (S: Sarakhs, B: Bane baghi, A: Abareqi). Bars indicate standard error. Colomns with different letters aresignificantly different at P< 0.05.
Figure 5. Effects of mycorrhizal symbiosis (M: with mycorrhizae, NM: without mycorrhizae) on leaf proline levels in three pistachio rootstocks (S: Sarakhs, B: Bane baghi, A: Abareqi) grown 70 days under different salt intensities (EC of 0.5, 5, 10 and 15 dS m-1). Bars indicate standard error. Colomns with different letters aresignificantly different at P< 0.05.
Figure 6. Effect of different salt levels (EC of 0.5, 5, 10 and 15 dS m-1) on root proline content of three pistachio rootstocks (S: Sarakhs, B: Bane baghi, A: Abareqi) 70 days after salt stress commencement. Bars indicate standard error. Colomns with different letters aresignificantly different at P< 0.05.
100 90 80
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Figure 7. Interaction effects of mycorrhizal symbiosis and salt intensities (EC of 0.5, 5, 10 and 15 dS m-1) on root proline contentof pistachio rootstocks. Bars indicate standard error. Colomns with different letters aresignificantly different at P< 0.05.
120
0.5 5 10 15
■ S B ■ A
Figure 8. Effect of salinity levels (EC of 0.5, 5, 10 and 15 dS m-1) on mycorrhizal root colonization percentage of three pistachio rootstocks (S: Sarakhs, B: Bane baghi, A: Abareqi) 70 days after salt stress commencement. Bars indicate standard error. Colomns with different letters aresignificantly different at P< 0.05.
Table 1. ANOVA results for mycorrhizal infection percentage(MI), leaf methylglyoxal (LMG), root methylglyoxal (RMG), leaf proline (LPRO) and root proline (RPRO)cotentof Abareqi, Sarakhs and Bane baghi rootstocks (PR) exposed to varying intensities of salt stress (S) andAMF (M) treatments.
Parameters M S PR MxS MxPR SxPR MxSxPR
MI% - *** *** - - *** -
LMG *** *** ns ** ** ns ns
RMG *** *** *** ns ns * ns
LPRO *** *** *** *** ns *** **
RPRO ns *** *** ** ns * ns
*** -significant (P<0.001); ** - - significant (P<0.01), * I g 3 c Qi 3 ( < o .05); ns - not significant.
Table 2. Relationship between the biomarker (Y) and salt stress (x) levels and their respective correlation coefficients for mycorrhizal and non-mycorrhizal pistachio plants.
Mycorrhizal treatments
Biomarkers -M +M
LMG Y= 17.7X + 76.12 Y=12.7X + 65.53
R2= 0.98*** R2= 0 99***
RMG Y= 5.4X + 31.2 Y= 4.25X + 27.55
R2= 0.96*** r2= 0.94***
LPRO Y= 12.89X + 14.44 Y= 7.9X + 18.48
R2= 0.91*** R2= 0.83**
RPRO Y= 4.62X + 18.59 Y= 3.59X + 23.78
R2= 0.98*** R2= 0 99***
DISCUSSION seems that increased levels of MG could act as a
In an attempt to determine whether the signal for plants to respond to the stress (Yadav et
accumulation of MG and proline in leaf and root al., 2005; Singla-Pareek et al., 2006). However, very
tissues of three different pistachio rootstocks, pre- little works have been done in plant systems
treated with mycorrhiza, in response to various salt regarding the endogenous production of MG.
stress levels is a common phenomenon, MG and Results presented in Fig.2 showed that leaf MG
proline levels were measured in this study. level in non-mycorrhized cv.Sarakhs seedlings was
Significant increase of MG levels were observed due highest while the lowest values were recorded for
to different concentration of NaCl that is in agree the mycorrhized seedlings of the same rootstock.
with previous studies (Yadav et al., 2005). The The opposite results were observed in cv. Abareqi.
sharper increase were observed in leaf tissues It can be attributed to the mycorrhization extent of
where cells become metabolically active under these rootstocks under different salinity levels since
stress condition which is mirrored by upregulation root colonization of Abareqi seedlings was reduced
of enzymes involved in glycolysis and TCA cycles severely as the effect of salinity whereas no obvious
(Sommer et al., 2001) and as a result, flux of triose reduction occurred in cv. Sarakhs under the same
phosphates increases which, instead of giving only conditions.
pyruvate could be converted to MG. Previously, It Leaf and root proline content was increased with
has been reported that MG accumulation in rice increase in the level of salinity. The increase of
roots is lower than shoots under salt stress (Yadav proline level in roots was less and gradual in
et al., 2005) which is confirmed by our results. It compare with leaves which is in agree with previous
results (Sofo et al., 2004). Abareqi possessed maximum values for leaf proline content at salinity levels of S3 and S4 compared to the others. Proline acts as a cytosolic osmoticum, scavenger of OH-radical and can interact with cellular macromolecules, such as DNA, protein, membranes, and can stabilize their structure and function (Kishor et al., 2005). Therefore, it was expected that the exposure of pistachio plants to NaCl could higher the level of proline in order to overcome the oxidative stress generated by the salinity. A possible reason for this increased level of proline during the salt stress could be an alteration in the activities of the enzymes involved in the biosynthesis and degradation of proline.
We found a very strong relationship between salinity and measured biochemical markers (Table 2). Significant positive correlation between leaf MG level and salt stress intensity in -M (R2 = 0.98***) and +M (R2 = 0.99***) pistachio rootstocks and between root proline and salinity levels in -M ((R2 =
0.98***) and +M (R2 = 0.99***) plants revealed that both of them could be used as a biochemical marker of salt stress level in pistachio plants.
The fact that under S4, cv. Sarakhs had the highest and lowest accumulation of leaf MG and proline (Fig. 2, 5) respectively and also cv. Abareqi had the highest and lowest of root proline and MG respectively under S3 and S4 could be related to the role of proline in enhancing MG detoxification systems (Hossain and Fujita, 2010).
In our experiment, root colonization extent of all rootstocks were not changed up to EC of 5 dS m-1 and thereafter, it was reduced in Abareqi and Bane baghi while did not change in Sarakhs (Fig. 8). Salinity, not only affects the host plant but also the AMF. It can hamper colonization capacity, spore germination and growth of hyphae of the fungus.
Several researchers have reported the negative effects of salinity on the fungus (Jahromi et al., 2008). A few studies reported that AMF colonization is not reduced in the presence of NaCl (Levy et al., 1983). Increased AMF sporulation and colonization under salt-stress conditions has also been reported (Aliasgharzadeh et al., 2001). However, unchanged colonization percentage in cv. Sarakhs under salt stress remains ambiguous, although it can be attributed to the quantity and quality of root exudates (Giovannetti et al., 1996), more research and understanding are needed to clarify its accurate mechanism.
ACKNOWLEDGEMENTS
This research was part of M.Sc. thesis of the second author. Financial support by Vali-e-Asr University of Rafsanjan is greatly acknowledged.
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