Oryza sativa L.

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Material and methods. Plant material. Four Iranian local and improved rice cultivars (Hassani and. Anbarboo as local; Nemat and Khazar as improved varieties),.
Different physiobiochemical and transcriptomic reactions of rice (Oryza sativa L.) cultivars differing in terms of salt sensitivity under salinity stress Mojtaba Kordrostami, Babak Rabiei & Hassan Hassani Kumleh

Environmental Science and Pollution Research ISSN 0944-1344 Environ Sci Pollut Res DOI 10.1007/s11356-017-8411-0

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Author's personal copy Environ Sci Pollut Res DOI 10.1007/s11356-017-8411-0

RESEARCH ARTICLE

Different physiobiochemical and transcriptomic reactions of rice (Oryza sativa L.) cultivars differing in terms of salt sensitivity under salinity stress Mojtaba Kordrostami 1 & Babak Rabiei 2 & Hassan Hassani Kumleh 1

Received: 15 July 2016 / Accepted: 5 January 2017 # Springer-Verlag Berlin Heidelberg 2017

Abstract Salinity stress is the most important and common environmental stresses throughout the world, including Iran. The aim of this study was to investigate the expression of several important genes involved in the salinity tolerance of the rice cultivars differing in salt sensitivity. In this research, the expression of four mitochondrial genes, H 2 O 2 , malondialdehyde (MDA), proline, sodium, potassium and superoxide dismutase (SOD), was measured in Iranian rice cultivars and two well-known international varieties as checks in response to 100 mM salt stress. The results show that the activity of SOD in the tolerant cultivars is much higher than in the susceptible ones under saline conditions (100 mM NaCl). The study of the gene expression in the tolerant and sensitive cultivars also showed that the expression of the genes increased in the early hours of the stress, with the exception of the OsGR1. Moreover, the amount of the expression in the tolerant cultivars was far more than the susceptible Responsible editor: Yi-ping Chen Electronic supplementary material The online version of this article (doi:10.1007/s11356-017-8411-0) contains supplementary material, which is available to authorized users. * Babak Rabiei [email protected] Mojtaba Kordrostami [email protected] Hassan Hassani Kumleh [email protected] 1

Department of Plant Biotechnology, Faculty of Agricultural Sciences, University of Guilan, P.O. Box: 41635-1314, Rasht, Iran

2

Department of Agronomy and Plant Breeding, Faculty of Agricultural Sciences, University of Guilan, P.O. Box: 41635-1314, Rasht, Iran

ones. The result of this study showed that the function of a set of antioxidant enzymes can lead to detoxification of the reactive oxygen species, so in order to better understand ROS scavengers, a comprehensive study on the antioxidant system should be conducted. Keywords Antioxidant genes . Glutathione peroxidase . H2O2 . NaCl . Superoxide dismutase

Introduction Rice belongs to the Poaceae or Gramineae family and the Oryza genus and contains 20 different species in which only 2 species, O. sativa and O. glaberrima, are cultivated. Today, the cultivation of O. sativa species has increased due to its high yield (Wopereis et al. 2009); this crop plant is also the second most important cereal in the world after the wheat. Natural environments possess the biotic and abiotic stresses for the plants. Plants are constantly exposed to the environmental changes and if these changes have much intensity and speed, they will be considered as the stress (Ciarmiello et al. 2011). In the FAO’s report, it is mentioned that only 5.3% of all the world’s land area is not influenced by the environmental stresses (FAO 2007). Cramer et al. (2011) defined the stress as the unfavourable environmental factors for the living organisms and resistance as the ability of the organisms to continue living in these unfavourable conditions. However, for the crop plants, the yield is as important as the plant survival (Cramer et al. 2011). Salinity (after the drought) is the most important and common environmental stress throughout the world, including Iran. A significant portion of the world’s natural and agricultural ecosystems are under salt stress. In Iran, about 25% of the land area is saline (Choukr 1996). Nowadays, due to the excessive use of natural resources and

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the use of inappropriate methods for the production of agricultural products, particularly in relation to the irrigation water, a significant portion of the agricultural lands in the arid areas are faced with saline soils (Puri and Williams 1985). In Iran, salinity is an inclusive problem which limits the sustainable agricultural production, so that large parts of the arid and semi-arid regions of the country (particularly the central plateau, southern coastal plains and the plain of Khuzestan) are suffering from different salinity levels (Momeni 2010). Due to the geographical position of Iran, most of its regions are arid and semi-arid. In this country, there are some regions whose rate of evaporation is eight times more than the rainfall. Therefore, using low-quality water, such as saline water, for crop production is not unexpected in most parts of the country (Kamali et al. 2011). In Iran, due to large amounts of salt deposits in the shallow depth of the soils, there is an abundance of salinity in the soils and groundwater; besides that, the secondary salinity happens and has been reported repeatedly (Van Weert et al. 2009). Despite the magnitude and the extent of the salinity problem, the government has not considered a comprehensive plan to control salt stress (Momeni 2010). The complex responses of the plants to the abiotic stresses are associated with the expression of multiple genes and different biochemical and molecular pathways. Many of these genes are co-expressed in various stresses. For example, many of the genes involved in the salt and cold stress are also coexpressed in drought stress, indicating the similar mechanisms which are involved in the response of these stresses (Cramer et al. 2011). Theoretically, the salt stress in the plants increases ROS production. At first, the plants reduce the stomatal conductance in the response of the salt stress to prevent the excessive loss of water. This can reduce the internal CO2 concentration (Ci) and cause the slower reduction of CO2 by the Calvin cycle. This response led to the evacuation of oxidized NADP+ (which works as a final acceptor of electrons in the PSI), the increase of the electron leakage to O2 and the formation of O2−. In addition, the toxicity of Na+/Cl− induced by the salt stress can disrupt the photosynthetic electron transfer and stimulate the electron leakage to O2 (Slesak et al. 2002). Secondly, a reduction in the internal CO2 concentration decreases the Calvin cycle reaction rates and induces photorespiration, especially in C3 plants. As a result, a large amount of H2O2 is produced in the peroxisomes. Thirdly, the membranebound NADPH-oxidase and apoplastic diamine-oxidase are activated during salt stress and so will assist in the construction of ROS (Lin and Kao 2001; Hernandez et al. 2003). Finally, salinity increases the respiration rate and therefore causes the respiratory electron leakage to O2 (Fry et al. 1986; Moser et al. 1991; Jeanjean et al. 1993). Superoxide dismutase (SOD) is one of the main scavenging enzymes which convert O2− to H2O2 and then coordinate the action of four enzymes including catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX) and

proxy-redoxins (Prxs) and induce H2O2 to O2 and H2O. Given the importance of salt stress and its role in crop performance reduction, engineering the resistance or tolerance to the salinity in the plants, has extremely important economic benefits. So, investigating the molecular mechanisms that are effective in the salinity tolerance is very important. The main strategy for the genetic engineering of the salt tolerance is the induction of the genes that are directly involved in these events (Mittler et al. 2004). In this research, the Iranian local and improved rice cultivars with two standard susceptible and resistant cultivars were evaluated under saline conditions (100 mM NaCl). The aim of this study was to investigate the expression of several important genes involved in the salinity tolerance in the rice cultivars and study their behaviour in the tolerant and susceptible cultivars under the severe salinity conditions.

Material and methods Plant material Four Iranian local and improved rice cultivars (Hassani and Anbarboo as local; Nemat and Khazar as improved varieties), along with two control-tolerant and sensitive cultivars (FL478 and IR29) were evaluated for different physiobiochemical and transcriptomic characteristics under salinity stress (100 mM NaCl). It should be noted that these cultivars were previously screened for salinity tolerance by Kordrostami et al. (2016). The research was conducted at the seedling stage of the studied varieties at the Biotechnology laboratory of Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran. To perform the experiment, a factorial analysis in a completely randomized design was used with three replications. The characteristics of the studied cultivars are presented in Table 1. Stress treatment In order to the disinfect the seeds and avoid the possible infections, firstly, the required seeds were fully washed with the distilled water and then they were placed in a 5% sodium Table 1

The studied rice genotypes

No. Cultivar

Origin

Reaction to salinity

G1 G2 G3 G4 G5 G6

IR66946-3R-178-1-1 Iranian landrace (Guilan) Amol3/Sangetarom Iranian landrace (Khouzestan) IR36/TNAU4756 IR833-6-2//IR1561-149-1//IR24*4/ON

Tolerant Tolerant Tolerant Susceptible Susceptible Susceptible

FL478 Hassani Nemat Anbarboo Khazar IR29

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hypochlorite solution for 20 min. At the end of this period, the seeds were disinfected three times, each time for 5 min, washed with the distilled water and then they were transferred to the petri dishes. After being sterilized, the seeds of each cultivar were transferred to the petri dishes containing sterile filter papers and for the germination, they were placed in the growth chamber with a constant temperature of 28 °C. After 6 days, the cultivation of the plantlets was done in the Yoshida nutrient solution in hydroponic containers at a constant temperature of 25 °C; 14 h of light and 10 h of darkness. During the growth period, the ambient temperature remained constant between 24 and 30 °C. After 20 days, the seedlings were exposed to 100 mM NaCl. The sampling for evaluation of the gene expression, MDA, proline, H2O2, SOD, K and Na were applied in the six stages, including 3, 6, 12, 24, 48 and 72 h after the NaCl treatment.

Estimation of H2O2, MDA and proline content H 2 O 2 determination was done according to Jana and Choudhuri (1981). To determine the H2O2 content, 3 ml of extracted solution was mixed with 1 ml of 0.1% (m/v) titanium chloride in 20% (v/v) H2SO4; then, the mixture was centrifuged at 6000×g for 15 min and the supernatant absorbance was measured at 410 nm. The thiobarbituric acid (TBA) reaction cited in Abdel Latef and Tran (2016) was used to determine the MDA content in the fresh root samples. The absorbance was measured at 532, 600 and 450 nm. The method described by Bates et al. (1973) was used to estimate root proline content, and the absorbance was measured at 520 nm using toluene as blank.

Statistical analysis Triplicate biological replications were used for the analysis of each parameter. Data were presented as means and theirs standard errors. Mean comparison among different treatments was done by analysis of variance analysis (ANOVA), and the significant differences were reported at p < 0.05 or 0.01. RNA extraction, cDNA synthesis and real-time PCR conditions The total RNA was extracted from the roots of the seedlings using Cinnagen RNA extraction kit (RNX-Plus Solution). The cDNA synthesis was performed according to Fermentas kit. The quality and quantity of the extracted RNAwere controlled using agarose gel electrophoresis and spectrophotometry, respectively. The Ubiquitin gene was used as the internal reference gene. Sequences of the specific gene primers are listed in Table 2. The size of the PCR products varied between 98 and 189 bp, and the melting temperature varied between 52.4 and 58.8 °C according to (G + C) percentage and the length of bands. The real-time PCR conditions were as follows: initial denaturation at 95 °C for 3 min, then at 95 °C for 10 s, annealing at 52.4 and 58.8 °C (depending on primer used) (Table 2) for 20 s, extension at 72 °C for 15 s and final extension at 72 °C for 7 min. Each sample was analysed in triplicate and the average of Ct values was calculated. To reveal the absence of contamination or primer dimmers, a non-template control (NTC) reaction with each primer pair was run. The 2−△△CT method was used for the quantitative analysis (Livak and Schmittgen 2001).

Results SOD activity The super oxide dismutase (SOD) activity was measured according to Giannopolitis and Reis’s (Giannopolitis and Ries 1977) method. The reaction mixture contained 0.1 mM EDTA, 50 mM phosphate (pH 7), 13 mM L-methionine, different NBT concentrations (60, 75, 90, 105, 120, 135 and 160 μM), 10 μL of enzyme extract and 0.12 mM of riboflavin. The formation of formasan was held in the Eppendorf tube (2 ml). For each probe, one tube was retained in the darkness. The others were illuminated by white light lamp (40-W fluorescent lamp) for 15 min at 20 °C. The absorption was measured at the wave length of 560 nm using spectrophotometer (Shimadzu, Kyoto, Japan) (Sakhno et al. 2014). The SOD activity was calculated using the following equation:   ðOD control−OD sampleÞ 100−  100 OD control SOD activity ¼ 50

The study of superoxide dismutase activity in non-stress conditions shows that the highest enzyme activity in the tolerant cultivar FL478 was observed at 48 and 72 h while the minimum activity was observed at 6 and 12 h (Supplemental Figs. 1a and 2a). In the sensitive cultivar IR29, however, this enzyme had the most activity in the early hours of the experiment (Supplemental Figs. 1b and 2a). Under salt stress conditions (100 mM NaCl), FL478 had the highest SOD activity at 3 and 72 h, while the minimum activity was observed at 6 and 12 h after the stress (Fig. 1a and Supplemental Fig. 3a). On the other hand, in the sensitive cultivar IR29, this enzyme had the most activity at 6 and 24 h after the stress (Fig. 1b and Supplemental Fig. 3a). By comparing superoxide dismutase activity in both stress and non-stress conditions in FL478, it can be concluded that the salinity caused a significant increase in the enzyme activity in this cultivar. However, it should not be forgotten that a variety of enzymatic and nonenzymatic mechanisms are involved in the tolerance or

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Specifications of the primers used in real-time PCR

Gene

Primer sequence

TM (°C)

PCR product (bp)

NCBI accession number

Ubq-F Ubq-R OsGPX1-F OsGPX-R OsAOX1c-F OsAOX1c-R OsUCP1-F OsUCP1-R OsGR1-F OsGR1-R

5′-ACCACTTCGACCGCCACTACT-3′ 5′-ACGCCTAAGCCTGCTGGTT-3′ 5′-AGCAACCTGCACTTATGCACT-3′ 5′-CAGCAAGGAAATTTATTGACATGA-3′ 5′-GAGTCTCCAGCTCGACCTC-3′ 5′-CTCCCGGTGTTCTCCTTGG-3′ 5′-ATCGCCTTGTATGAGCCTGT-3′ 5′-CTTGACAGTGGTGGCCTTTC-3′ 5′-GCACACCAAACTCCCAGAGG-3′ 5′-CCTTGGTGGCTCCACACTTC-3′

54

157

XM_006644067.1

52.4

189

AY100689.1

59.9

98

AB074005.1

58.4

103

AB049997.1

58.8

153

XM_015771322.1

sensitivity of a genotype to oxidative stress. Interestingly, under salt stress conditions, SOD activity increased in IR29. However, it should be stated that the enzyme activity was Fig. 1 The reaction rate of SOD at different times in different substrate concentrations under salt stress conditions: a FL478, b IR29, c Hassani, d Anbarboo, e Nemat, f Khazar

much higher in the tolerant cultivar than in the susceptible one; this factor may somehow explain the tolerance of FL478 rather than IR29.

Author's personal copy Environ Sci Pollut Res Fig. 2 qPCR analysis of the expression profiles of OsGPX1 gene during salt stress in the rice seedlings. Gene expression in: a FL478 and IR29, b Hassani and Anbarboo, c Nemat and Khazar. The error bars represent standard deviation among the biological replicates

The study of the superoxide dismutase activity in Hassani (Iranian local salt tolerant cultivar) under non-stress conditions demonstrated that the highest enzyme activity was observed at 3 h, but the amount of the enzyme immediately had a remarkable drop and began to increase again, continuing until the late hours of the experiment (Supplemental Figs. 1c and 2b). However, superoxide dismutase activity in Anbarboo (Iranian local salt sensitive cultivar) did not follow a specific pattern. Anbarboo had the highest SOD activity at 24 and 72 h, while minimum activity was observed at 3 to 12 h (Supplemental Figs. 1d and 2b). Therefore, we cannot find a proved pattern for this enzyme in non-stress conditions. Under salt stress conditions (100 mM NaCl), Hassani had the highest enzyme activity at 3 and 48 h after stress (Fig. 1c and Supplemental Fig. 3b). In this cultivar, the lowest activity of the enzyme was observed near the end of the stress period. To some extent, the superoxide dismutase activity in Anbarboo followed the same pattern as Hassani (Fig. 1d and Supplemental Fig. 3b)—it had the highest enzyme activity at 3 and 24 h after the stress. In Hassani, a comparison of superoxide dismutase activity in both stress and non-stress conditions indicates that salinity caused a significant increase in the enzyme activity in this cultivar; this can explain the relatively

higher tolerance of Hassani, compared to Anbarboo, under salt stress conditions. The study of superoxide dismutase activity in Nemat (under non-stress conditions) showed that the maximum activity for this enzyme was observed at 24 h (Supplemental Figs. 1e and 2c). Khazar (under non-stress conditions) also showed the same pattern in SOD activity (Supplemental Figs. 1f and 2c). The highest activity of this enzyme was observed at 3 h. Under salt stress conditions, these two varieties followed the same pattern as FL478 and IR29. Under salt stress conditions (100 mM NaCl), Nemat had the highest SOD activity at 6 and 72 h, while minimum activity was observed at 12 and 24 h after the stress (Fig. 1e and Supplemental Fig. 3c). In the sensitive cultivar Khazar, this enzyme had the most activity in the early hours of stress; however, its activity did not fall over time and the highest activity was recorded at 48 and 72 h after the stress (Fig. 1f and Supplemental Fig. 3c). By comparing the superoxide dismutase activities in the two conditions, it can be stated that enzyme activity in Khazar was higher than that in Nemat. On the other hand, by comparing the activity of this enzyme in Khazar in the two conditions, it can be concluded that the level of superoxide dismutase activity decreased on inducing the stress. This can explain the

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relative sensitivity of Khazar compared to Nemat under salt stress conditions. The changes in the proline content of the cultivars during the stress period are shown in Supplemental Fig. 4. As can be seen, the proline content was almost variable for the different times and different varieties. For example, in FL478, the proline increased with a gentle slope up to 24 h after the stress (100 mM NaCl). After that, the slope had a noticeable increase. However, in the two tolerant Iranian cultivars (local and improved), the situation was slightly different. For example, in Hassani, the proline had an increasing slope up to 12 h after the stress. The level did not change up to 24 h, and then decreased to an extent. Such a situation was also observed for Nemat. In Nemat, the proline had an increasing slope up to 12 h after the stress. After that, it did not change up to 48 h and then it decreased. But, the behaviour of proline was completely variable in the susceptible cultivars (Supplemental Fig. 4). For example, in IR29, the proline content increased up to 72 h after the stress, even though the proline content in this cultivar was far less than that in the tolerant one. In Anbarboo, the proline content increased up to 24 h after the stress and then its value decreased. Among the susceptible cultivars, Khazar did not follow any specific pattern. In this cultivar, we observed an intermittently increasing and decreasing trend in the proline content during the stress period. The changes in the H2O2 content of the cultivars during the stress period (100 mM NaCl) are shown in Supplemental Fig. 5. As can be seen, the H2O2 content was almost variable at the different times and in the different varieties. For example, in FL478, the H2O2 increased with a gentle slope up to 6 h after the stress but had a significant decrease over time after that. In two resistant Iranian cultivars (local and improved), the situation was slightly different. For example, in Hassani, the H2O2 increased with a gentle slope up to 12 h after the stress but had a significant decrease over time after that. Nemat had a different situation; in this cultivar, the H2O2 had an increasing slope up to 12 h after the stress but then did not change up to 24 h; after that, some decrease was observed. But, the behaviour of H2O2 was mostly variable for the susceptible cultivars shown in Supplemental Fig. 3. For instance, in IR29, the amount of H2O2 increased up to 48 h after the stress; at 72 h, it declined mildly. In Anbarboo, H2O2 increased with a gentle slope up to 24 h after the stress, but after that (at 48 h), it had a significant decrease over time. Among the susceptible cultivars, Khazar did not follow any specific pattern. In this cultivar, we observed an increase in the amount of H2O2 from the early hours up to the final hours of the stress. The mean comparison results for MDA content are presented in Supplemental Fig. 6. The results show that in all the studied cultivars, the amount of MDA increases over time. However, the amount of the MDA in the tolerant genotypes was far less than that in the sensitive ones. The highest amount

of malondialdehyde (72 h after the stress) was observed in Khazar, Anbarboo and IR29 cultivars, respectively (Supplemental Fig. 6). The six studied genotypes were different in terms of the rate of sodium and potassium accumulation in saline conditions (100 mM NaCl) (Supplemental Fig. 7). The results of this study show that the tolerant cultivars (FL478, Hassani and Nemat) accumulated a smaller amount of Na+ (and higher levels of K+) in their roots and had more favourable Na+/K+ ratio compared with IR29, Khazar and Anbarboo under salt stress conditions. Also, the amount of K+ was decreased in all the genotypes in response to salt stress; this decrease in sensitive genotypes was more than that in the tolerant ones (Supplemental Fig. 7). The study of glutathione peroxidase gene expression (OsGPX1) in FL478 (the tolerant cultivar) shows that the highest expression was observed 6 h after the stress. Then, in the time period of 12–72 h after the stress, the expression of this gene decreased significantly (Fig. 2a). OsGPX1 expression in IR29 (the sensitive cultivar) first increased and then decreased by increasing the duration of the stress (Fig. 2a). In this way, 6 to 12 h after the stress, it was at the highest level (however, the expression of this gene was negligible in the same period compared to the FL478), and then decreased again. At 72 h after the stress, it reached the minimum level. The comparison of the gene expression in both the tolerant and the susceptible control cultivars showed that although this gene was expressed in both genotypes in the early hours of the stress, the expression of this gene was far more in the tolerant genotype than in the susceptible one. The study of glutathione peroxidase gene expression (OsGPX1) in Hassani and Anbarboo (two local tolerant and sensitive cultivars from Iran) showed that these cultivars followed the same pattern as the control varieties (Fig. 2b). Therefore, the highest expression of this gene in Hassani was observed 6 h after the stress. Then, in the time period of 12–72 h after the stress, the expression of this gene decreased significantly. GPX1 expression in Anbarboo (the local sensitive genotype) first increased and then decreased by increasing the duration of the stress (Fig. 2b). In this way, 3 to 12 h after the stress, it was at the highest level and then decreased again. At 72 h after the stress, it reached its minimum level. The study of glutathione peroxidase gene expression (OsGPX1) in Nemat and Khazar (two improved tolerant and sensitive cultivars from Iran) showed that the highest expression was observed 6 h after the stress. Then, in the time period of 12–72 h after the stress, the expression of this gene significantly decreased. The important point in this regard is the increased expression of this gene in Khazar compared to Nemat (Fig. 2c). Alternative oxidase gene expression (OsAOX-1c) in FL478 showed that the expression of this gene does not change much in the late period of the stress, but the expression of this gene significantly increased in the early hours of the stress (3 and

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12 h) (Fig. 3a). In IR29, the highest expression of the gene was observed at 12 h after the stress. Then, in the time period of 12–72 h after the stress, the expression of this gene remained unchanged (Fig. 3a). Based on the observations, although this gene was expressed in the early hours in both the susceptible and the resistant cultivars, the expression of this gene in the tolerant cultivars was far higher than in the sensitive ones. In one sense, it should be noted that the relative expression of this gene was much higher in these two varieties than in the improved varieties in the same situation. Alternative oxidase gene expression (OsAOX-1c) in Nemat showed that the expression of this gene did not change much in the late period of the stress but significantly increased in the early hours of the stress (6 and 12 h) (Fig. 3c). The study of OsGR1 gene expression in FL478 showed that the expression of this gene increased over time. The highest amount was observed at 48 h after the stress. Then, 72 h after the stress, its expression significantly decreased (Fig. 4a). In IR29, the expression of this gene increased over time and then reduced. In this way, at 24 and 48 h after the stress, it was at the highest level (however, the expression of Fig. 3 qPCR analysis of the expression profiles of OsAOX1 gene during salt stress in the rice seedlings. Gene expression in: a FL478 and IR29, b Hassani and Anbarboo, c Nemat and Khazar. The error bars represent standard deviation among the biological replicates

this gene was negligible compared to the FL478 in the same period) and then decreased (Fig. 4b). The comparison of the gene expression in both tolerant and susceptible control cultivars showed that although this gene was expressed in both the genotypes in the late hours of the stress, the expression of the gene in the tolerant genotype was far more than that in the susceptible one and the differences were significant. The study of OsGR1 gene expression in Hassani showed that the expression of this gene increased over time. The highest amount was observed at 48 h after the stress (Fig. 4b). In Anbarboo, the expression of this gene increased over time and then decreased. In this way, at 24 and 48 h after the stress, it was at the highest level (however, the expression of this gene was negligible compared to the FL478 in the same period) and then decreased again at 72 h after the stress (Fig. 4b). The study of OsGR1 gene expression in Nemat showed that the expression of this gene increased over time. The highest amount was observed at 48 h after the stress. Then, 72 h after the stress, its expression significantly decreased (Fig. 4c). However, in Khazar, the expression of the gene followed an ascending and descending trend (Fig. 4c).

Author's personal copy Environ Sci Pollut Res Fig. 4 qPCR analysis of the expression profiles of OsGR1 gene during salt stress in the rice seedlings. Gene expression in: a FL478 and IR29, b Hassani and Anbarboo, c Nemat and Khazar. The error bars represent standard deviation among the biological replicates

The study of OsUCP1 gene expression in FL478 showed that the highest expression was observed 6 h after the stress. Then, in the time period of 12–72 h after the stress, the expression of this gene significantly decreased (Fig. 5a). OsUCP1 expression in IR29 (the sensitive genotype) first increased and then decreased by increasing the duration of the stress. In this way, 6 to 12 h after the stress, it was at the highest level (however, the expression of this gene was negligible compared to the FL478 in the same period) and then decreased again. At 72 h after the stress, it reached the minimum level (Fig. 5a). The study of uncoupling protein (OsUCP1) gene expression in Hassani and Anbarboo (two local tolerant and sensitive genotypes from Iran) showed that these cultivars followed the same pattern as the control varieties (Fig. 5b). Therefore, the highest expression of this gene in Hassani was observed 6 h after the stress. Then, in the time period of 12–72 h after the stress, the expression of this gene significantly decreased. However, 24 h after the stress, the expression of this gene increased mildly. OsUCP1 expression in Anbarboo (the local sensitive genotype) first increased and then decreased by increasing the duration of the stress. In this way, 3 to 12 h after the stress, it was at the highest level and then decreased again. At 72 h after the stress, it reached its minimum level. The study of glutathione peroxidase gene expression (OsUCP1) in Nemat and Khazar (two improved

tolerant and sensitive cultivars from Iran) showed that the highest expression was observed 6 h after the stress. Then, in the time period of 12–72 h after the stress, the expression of this gene significantly decreased (Fig. 5c). The important point in this regard was the increased expression of this gene in Khazar compared to Nemat.

Discussion In this study, the activity of ROS scavengers was studied under salt stress conditions (100 mM NaCl). The results showed that SOD activity rose sharply under salt stress in all the genotypes. In the present study, the tolerant cultivars had a greater amount of superoxide dismutase. The Supplemental Table 1 shows that SOD (normal conditions) did not have any correlation with any parameters. On the other hand, there was a negative correlation between SOD (salt stress conditions), Na, H2O2 and MDA content. There was also a positive correlation between SOD (salt stress conditions), proline and tolerance. It shows that the tolerant genotype had more SOD activity and proline content and less MDA, Na and H2O2 content. The results of our study show that the SOD activity increased or remained unchanged in the early stages of salt stress but was elevated by long-term salt stress. Our results

Author's personal copy Environ Sci Pollut Res Fig. 5 qPCR analysis of the expression profiles of OsUCP1 gene during salt stress in the rice seedlings. Gene expression in: a FL478 and IR29, b Hassani and Anbarboo, c Nemat and Khazar. The error bars represent standard deviation among the biological replicates

were in line with Abbasi and Komatsu (2004). The results of the correlation analysis showed that there is a positive correlation between SOD and the expression of OsGPX, OsGR and OsAOX. As in our study, Joseph et al. (2015) demonstrated that antioxidants such as glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxiredoxin (POD) can provide oxidative stress resistance in rice. The need for higher levels of the SOD is found naturally in the halophytes. This is essential for expedite signalling of the presence of H2O2 and triggering an adaptive cascade response (genetic and physiological). Mishra et al. (2013) observed an increase in SOD activity and its isoforms (Cu/Zn-SOD) in both tolerant and sensitive rice varieties under salt stress conditions. Prior to this, it has been shown that salinity increased the SOD activity in the salt-tolerant cultivars and decreased its activity in the susceptible ones in both leaf and root tissues under salt stress conditions (Shalata et al. 2001). Our study shows a negative correlation between SOD and Na content. It shows that in the sensitive genotypes, a loss in the ability to detoxify free radicals is attributed to the reduced activity of antioxidant enzymes such as SOD (Mittova et al. 2002; Desingh and Kanagaraj 2007). In another study, it had been found that the salt-tolerant rice cultivars increased

the activity of superoxide dismutase and decreased the lipid peroxidation (Dionisio-Sese and Tobita 1998). In the present study, the relative expression of OsGPX1 was increased in the tolerant genotypes, which indicate the importance of this gene. There was also a strong negative c o r r el a t i o n b e t w e e n O s G P X 1 a n d M D A c o n t e n t (Supplemental Table 1). Due to the exclusive role of OsGPX1 in the direct reduction of phospholipidhydrogenase and hydroperoxyl complex in protecting cell membranes against the oxidative stress (hence, OsGPX1 protect cell membranes from the oxidative damage and thereby maintaining the cell integrity), the presence and extent of this gene in the different organs seem justifiable (Herbette et al. 2002; Jung et al. 2002; Navrot et al. 2006). The results show that there is a strong positive correlation between OsGPX1 and SOD content (Supplemental Table 1). SOD converts O2− to H2O2 and then coordinates the actions of four enzymes—catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX) and proxy-redoxins (Prxs). Finally, it induces the H2O2 to O2 and H2O. This is because there is a positive correlation between these two enzymes in the tolerant genotypes. There was also a positive correlation between the tolerance and OsGPX1 relative expression. In Fig. 2, it can be

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seen that the expression of the OsGPX1 did not change in the sensitive genotypes during the stress period. The results also showed that the expression of this gene in the more susceptible cultivars (IR29) were lower than in the other varieties. The results of this study show that the relative expression of OsGR1 in the tolerant genotypes was more than that in the sensitive ones. In Supplemental Table 1, it can be seen that there is a positive correlation between the tolerance and the OsGR1 expression. It shows that under salt stress conditions, the relative expression of this gene was higher than the sensitive ones. The results also showed that the expression of this gene did not change dramatically during the stress period in the sensitive genotypes. The results of the other studies also indicated that glutathione reductase plays an important role in oxidative stress adaptation in plants. These enzymes are responsible for the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) and maintaining the high GSH/ GSSG ratio. Glutathione plays an important role in xanthophyll, Mehler and ascorbate-glutathione cycles and hydrogen peroxide collection and preservation of GSH. GSH can act as an antioxidant (non-enzymatic) like the ascorbate and directly purify the singlet oxygen (O2) (Noctor and Foyer 1998). Studies have shown that GR and APX activity are increased in rice plants during the oxidative stress, which are in line with the results of this study (Sharma and Dubey 2005). In the present study, the salt treatment significantly increased the OsGR1 expression. Moreover, the researches show that the salt treatment could increase the activity of GR and have a significant effect on the activation of two GR isoenzymes (OsGR1 and OsGR2) in the roots of the rice plants (Tsai et al. 2004), which are in line with the results of this study. In this study, the OsGR1 gene expression increased in both the tolerant and the sensitive genotype under salt stress conditions. However, the expression of this gene was far greater in the tolerant genotype than in the susceptible one. In the present study, the relative expression of OsAOX1-c was increased in both the tolerant and the sensitive genotype. The evidence suggests that the AOX in the plants is encoded by the small multi-family genes and plays a significant role in the unfavourable environmental conditions in two ways: (1) adjustment of plant growth and development and (2) protection of the cells from the oxidative stress (Polidoros et al. 2009). The previous studies have shown that AOX1a and AOX1b genes have been induced by cold, drought and salinity in the roots and leaves of the rice seedlings, while it has been found that the AOX1c gene is not responsible for salt stress (Mittler et al. 2004). The results of this study are contrary to that of the previous studies. Our study showed that AOX1c gene expression was increased under salt stress conditions in both tolerant and sensitive genotypes. On the other hand, the results of our study showed that there is no correlation between tolerance and the AOX1c gene expression (Supplemental Table 1). This shows that both the tolerant

and the sensitive genotype had the same gene expression under salt stress conditions. However, the expression of this gene in the susceptible genotypes was far less than the tolerant ones in the same situation. These observations indicate that there are several qualitative and quantitative expressional patterns among different AOX genes under various abiotic stress levels. Therefore, a study that only examines changes in total protein or mRNA of AOX does not reflect the actual changes in the copies of different AOX genes under abiotic stresses (Mittler et al. 2004). One of the most common responses of the cell to the outside changes of osmotic pressure is the accumulation of the metabolites that have solubility but do not disrupt the normal metabolism of the plants (Orcutt and Nilsen 2000). Proline acts as a key osmoregulating solute in the cells under various stresses such as low temperature, food shortage, heavy metals and high acidity. In this study, the different genotypes follow the different patterns in the proline content. Large-scale proline production prevents the destructive effect of salinity on the natural processes of the cell (Paul and Hasegava 1996; Solomon and Beer 1994). Our study showed that proline content increased under salt stress conditions in both tolerant and sensitive genotypes. However, its content in the susceptible genotypes was far less than that in the tolerant ones in the same situation. There was also a positive correlation between the tolerance and the proline content (Supplemental Table 1), demonstrating its protective role against the destructive effect of salinity on the natural process of the cell in the tolerant genotypes. In the present study, the Na content increased in both the tolerant and the sensitive genotype during the stress period. However, in the same situation, its content in the susceptible genotypes was far more than in the tolerant ones. In the comparison, the K and K/Na ratio were in the opposite situation. The significant negative correlation between Na, K and K/Na ratio showed the antagonistic relationship between sodium and potassium in rice plants (Supplemental Table 1). This matter reduced the amount of absorbed potassium and the K/Na ratio. Our results are in line with those of Sabouri et al. (2008). Under salt stress conditions, by increasing the concentration of sodium in the plant, ion imbalance, nutrient deficiency and ion toxicity would occur. It is reported that the growth reduction of plants in high concentrations of sodium is due to the lack of potassium and calcium absorption (Mousa et al. 2013). Potassium is known to be an exchangeable ion in sodium excretion through the cell membrane system, because the excretion of sodium in the cortex is associated with the entry of potassium. The presence of a large quantity of sodium prevents the absorption of nutrients such as potassium in the plant tissues, which leads to an increase in the Na/K ratio (Kaouther et al. 2013). This is justifiable because the results showed that under salt stress conditions, the increase in the amount of sodium in plants and its accumulation in the

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cytoplasm enables the replacement of potassium with the sodium ions and leads to ion toxicity effects (Munns 2002). The results showed that there is a negative correlation between Na and tolerance (Supplemental Table 1). This shows that the tolerant genotypes hold a lower amount of sodium in their root tissues. Our results also showed that the tolerant varieties had high K/Na ratio. Our results were in line with those of other researchers (Ashraf and McNeilly 2004; López-Aguilar et al. 2012), who stated that the high K/Na ratio retention in the plants is essential for the plant salt tolerance and that cells need high K/Na ratio to perform their normal activities (Munns 2002; Azuma et al. 2010). In this research, under salt stress conditions, MDA content increased in all the genotypes. However, its increase in the susceptible genotypes was far more than the tolerant ones in the same situation. The results also showed that there was a negative correlation between tolerance and MDA, Na and H2O2 (Supplemental Table 1). It shows that the tolerant genotypes have the antioxidant system needed to reduce the H2O2 and MDA content. There was also a strong negative correlation between SOD, OsGPX1 and MDA (Supplemental Table 1). OsGPX1 is responsible for the inhibition of lipid peroxidation under oxidative stress. SOD is also a main enzyme for antioxidation. In this study, the increased salinity caused membrane lipid peroxidation, which reflects the damage caused by oxidative stress as a result of increased ROS. Free radicals increase the damage of the polar lipids in the membrane. Thus, oxidative stress leads to increased levels of lipid peroxidation under salt stress in the treated plants. MDA—the main decomposition product of unsaturated fatty acids in the biological membranes—is increased under salt stress conditions (Meloni et al. 2003; Sudhakar et al. 2001). MDA is regarded as a marker for the evaluation of lipid peroxidation or the damage of plasmalemmal and organelle membranes that increase due to environmental stress (Sudhakar et al. 2001). A significant increase in the levels of lipid peroxidation (MDA and other aldehydes) as an indicator that reflects the increasing damage of the membrane of the plants is observed under salt stress conditions (Stepien and Klobus 2005; Sudhakar et al. 2001). Lipid peroxidation is linked to the activity of antioxidant enzymes, e.g. SOD, APX, GPX, CAT, etc. By increasing such enzymes, oxidative stress tolerance will be enhanced and MDA will be decreased.

the cells causes damage to the cell membranes and to major macrocellular molecules such as RNA, DNA and vital enzymes. The results of this study show that the tolerant local and improved rice varieties follow a pattern to fairly similar FL478 in the SOD activity. The results show that the activity of SOD in the tolerant cultivars is much higher than in the susceptible ones under saline conditions (100 mM NaCl). The study of the gene expression in the tolerant and sensitive cultivars also showed that the expression of the genes increased in the early hours of the stress, with the exception of the OsGR1. Moreover, the amount of the expression in the tolerant genotypes was far more than that in the susceptible ones. The correlation analysis showed that the tolerant genotypes had a strong antioxidant system (proline, SOD, OsGPX, OsGR and OsUCP) and accumulated less Na, MDA and H2O2 in their root tissues. The results also showed that the sensitive genotypes failed to establish a balance between the antioxidant system and the stress factors. On the other hand, the results showed that there was a strong positive correlation between Na and H2O2. Apart from that, there was a strong negative correlation between H2O2 and the antioxidant system. It proves that salt stress stimulates the H2O2 production. Moreover, the plant antioxidant system in the tolerant genotype detects H2O2 and scavenges it in such a way that the plant cells are protected from destruction. The studies show that the majority of Iranian rice varieties are sensitive to salinity stress (Mohammadi-Nejad et al. 2010; Ghomi et al. 2013; Kordrostami et al. 2016). Considering this fact, the identification of the source of tolerance in the Iranian rice germplasm can help increase the salt tolerance in the high-yielding varieties. In this study, the high expression of the antioxidant genes related to the oxidative stress tolerance in the Iranian tolerant rice varieties shows that they can be induced in the Iranian high-performance salt-sensitive varieties using the new molecular techniques (genetic engineering), in order to adopt them to the Iranian complex environmental conditions. On the other hand, our results showed that the function of a set of antioxidant enzymes can lead to the detoxification of reactive oxygen species; thus, in order to better understand ROS scavengers, a comprehensive study of the antioxidant system should be conducted.

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Conclusion The production of oxygen-free radicals is one of the most destructive factors in the plants’ environmental stresses, which include salinity, drought and frost. Chloroplasts and mitochondria (the two major sites of electron transfer cycles in the plant cells) are always subject to the production of reactive oxygen species. The presence of reactive oxygen species in

Conflict of interest The authors declare that they have no competing interests. Research involving human participants and/or animals The authors declare that the present study does not involve any human participants and/or animals. Informed consent The authors declare that the present study does not involve any informed consent.

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