International Journal of Farming and Allied Sciences
Available online at www.ijfas.com
©2016 IJFAS Journal-2016-5-6/419-426/ 31 August, 2016 ISSN 2322-4134 ©2016 IJFAS
Salt tolerance determination in advanced mutant line of rice (Oryza sativa) at seedling stage
Parinaz Jalali, Saeid Navabpour
*, Ahad Yamchi, Hassan Soltanloo, Saeed Bagherikia
Department of Plant Breeding and Biotechnology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
Corresponding author: Saeid Navabpour
ABSTRACT: Salt stress imposes a major challenge to agricultural production, especially in arid and semi-arid regions. Rice (Oryza sativa) is the most salt-sensitive cereal crop. Therefore, the objective of this study was to investigate the effect of salinity on the plant growth trials and some osmoprotectants concentration in two rice genotypes (Hashemi parental and Hashemi advanced mutant line). For this purpose, seedlings were grown hydroponically under 0 (control), 5 and 10 dS m-1 NaCl stresses and different growth, qualitative and physiological traits were evaluated in three times including 6 h, 48 h and 1 w after salinity treatment. The results showed that salinity significantly reduced total fresh and dry weights as well as total chlorophyll content in both genotypes. Moreover, proline and trehalose content (as osmoprotectants) significantly increased in response to salinity stress. It was revealed that Hashemi advanced mutant line significantly delayed decreasing of total chlorophyll and had the highest proline and trehalose content as compared with Hashemi parental genotype. Overall, the Hashemi advanced mutant line that had the highest chlorophyll and osmoprotectants content identified as salt-tolerant genotype.
Keywords: chlorophyll, osmoprotectants, plant growth, proline, trehalose INTRODUCTION
Throughout their life, plants come across perturbations in environmental conditions which hamper their growth and development. In crop plants, such fluctuations, mostly in the form of soil salinity and drought, reduce yield (Tripathy et al., 2015). Salinity is a major environmental limiting factor that affects growth and productivity as well as the geographical distribution of many plant species. High salinity, predominantly in the form of NaCl, affects plant growth in three ways: osmotic effects, ion toxicity and nutrient imbalance. A high salt concentration in the rhizosphere impairs water uptake (osmotic effects) and nutrient absorption (nutrient imbalance) in plant roots.
However, excess accumulation of salt within the plant is highly toxic and results in oxidative stresses and enzyme inhibition (ion toxicity) (Munns and Tester 2008; Hosseini et al., 2015; Hossain et al., 2016).
The amount of salt-affected land has increased, and will continue to increase, due to continued unsustainable cultivation practices and climate change. A recent estimate claims that the annual cost of salt-induced land degradation in irrigated areas due to loss of crop production (Qadir et al. 2014).
Salt stress affects a wide variety of physiological and metabolic processes in plants in their vegetative stages leading to growth reduction. The response of plants to salinity is usually assessed by measuring the amount of biomass produced under saline and control conditions during long-term salt stress treatment (Munns 2002;
Hosseini et al., 2015). Shortly after exposure to salt stress, plants experience a cell growth and development reduction, which is largely caused by the salt outside the roots and is known as an osmotic effect (Munns 2002;
Munns and Tester 2008). Moreover, salinity frequently induces premature senescence of leaves. Leaf senescence
420 is most often quantized by decreases in protein or chlorophyll concentration and by increases in membrane permeability (Lutts et al., 1996).
Plants respond to salt stress by restricting the uptake of Na+, and adjustment of the cytoplasmic compartment is achieved by producing compatible osmolytes such as proline, ectoine, glycine betaine, sorbitol and trehalose. The high concentration of compatible solutes is able to balance the concentration of salts outside the cell on one side, and on the other, to counteract the high concentrations of Na+ and Cl− in the vacuole (Nounjan et al., 2012; Tu et al., 2014).
Proline is the most common osmolyte accumulating in plants in response to various stress conditions. It offers a wide range of protective roles including osmotic adjustment, stabilizer for cellular structure and reduction of damage to the photosynthetic apparatus. The level of Pro accumulation in plants varies from species to species.
The importance of Pro in enhancing plant stress tolerance has recently been substantiated through a transgenic approach (Su and Wu, 2004; Nounjan et al., 2012).
Trehalose is a non-reducing disaccharide found in many organisms. It is an essential component of the mechanisms that coordinate metabolism with plant growth adaptation and development (Paul, 2007). Trehalose accumulation influences the alteration of sugar metabolism leading to an osmoprotectant effect under stress (Djilianov et al., 2005).
Rice (Oryza sativa) feeds the human population more than any other crop, but it is a moderately salt sensitive crop in risk of greater exposure to brackish water due to the elevation of sea level, especially in the delta regions where rice is mainly produced (Pires et al., 2015). Rice could only tolerate salinities within the range of 1.9–3 dS/m and under salinities exceeding 6 dS/m it may experience up to 50% reduction in yield (Hashemi et al., 2016).
Due to the importance of rice as a global commodity, and the potential threat of increasing soil salinity to rice production, this study was conducted to study the effects of salinity stress at seedling stage on total biomass, chlorophyll content and some osmoprotectants concentration in two Hashemi parental and Hashemi advanced mutant line of rice under hydroponic conditions.
Materials and Methods
Plant materials and salinity experiment
This study was performed at the Faculty of Agriculture, University of Gorgan, Iran. Seeds of Hashemi parental and Hashemi advanced mutant line were obtained from the Rice Research Institute of Iran, Rasht. These genotypes were selected because they are some of the commonly grown rice cultivars in North of Iran and have high yield.
After breaking dormancy, seeds were surface sterilized with 5% (w/v) sodium hypochlorite for 15 min and rinsed four times thoroughly with distilled water prior to being sown in Petri dishes containing distilled water just sufficient to soak the seeds and incubated at 30 °C under dark condition for 5 days for uniform germination [8]. Five-day-old pre-germinated uniform seedlings (with 2-3 leaves) of the 2 rice genotypes were transplanted in foam-plugged holes (one plant per hole) in Styrofoam sheets floating over of Yoshida’s nutrient solution [9]. It is good for one month without replacement and maintenance of daily pH. The nutrient solution pH was checked every week for any deviation from 5.0 ± 0.5 (adjusted by adding either 1 N NaOH or 1 N HCl). The seedlings were grown in the phytotron facilities that are maintained at 29/21°C day/night temperature and at a minimum relative humidity of 70%. Twenty days after being transferred to the hydroponic system, plants were treated with 0 (as control), 5 and 10 dS m−1 of electrical conductivity (EC) by dissolving NaCl in the nutrient solution.
Morphological traits
To quantify and compare the responses of the contrasting genotypes under control and saline conditions, morphological parameters such as total fresh weight (FW) and dry weight (DW) were measured. Seedlings were harvested at three different stages including, 1) 6 h after treatment, 2) 48 h after treatment and 3) 1 week after treatment. Total FW was determined immediately after harvesting and total DW was determined after oven dried at 70 °C for 3 d.
Total chlorophyll content
The amount of total chlorophyll content was determined by UV-vis spectrophotometry as described by Porra et al.
(1989) and Holm (1954). Briefly, 10 mL of acetone 80% (acetone : distilled water 80:20 v:v) was added to 0.5 g of homogenized freeze-dried pulp leaves samples. Extract was then centrifuged at 1500 g for 10 min. The supernatant was separated and the absorbances were read at 663.6, 646.6 and 440.5 nm. The total chlorophyll content was calculated according to the following formulas:
Chl a = 12.25 A663.6 – 2.55 A646.6
421 Chl b = 20.31 A646.6 – 4.91 A636.6
Total Chl (mg g-1FW) = Chl a + Chl b Proline
Proline colorimetric determination proceeded according to Bates et al. (Bates et al., 1973). Briefly, 0.2 g of fresh leaf samples were homogenized in aqueous solution of 3% (w:v) sulphosalicylic acid, then centrifuged at 12000 g for 10 min. For proline determinations, the plant extract, acid ninhydrin and glacial acetic acid (2:2:2) were incubated at 100 °C for 1 h. The reaction was terminated in an ice bath. The reaction mixture was extracted with 4 mL of toluene and the chromophore-containing toluenephase was sucked. Proline content was measured spectrophotometrically at 520 nm using toluene as a blank and calculated as μmol g-1 FW against standard proline.
Trehalose
Trehalose content in the second leaves was determined following the method described by Mostofa et al. (2015) with some modifications. The leaves (1.0 g) were homogenized in 5 mL of 80% (v/v) hot ethanol and centrifuged at 11,500 × g for 20 min. The supernatants were dried at 80 °C followed by re-suspension in 5 mL distilled water. The solution (100 μ L) was mixed with 150 μL 0.2 N H2SO4 and boiled at 100 °C for 10 min to hydrolyze any sucrose or glucose-1-phosphate, then chilled on ice. NaOH (0.6 N, 150 μ L) was added to the above mixture and boiled for 10 min to destroy reducing sugars, then chilled again. Anthrone reagent (2.0 mL; 0.2 g anthrone per 100 mL of 95%
H2SO4) was added to the above mixture and boiled for 10 min to develop a color and then chilled again. The absorbance was recorded at 630 nm and Tre concentration was calculated as μg g−1 FW using a standard curve developed with commercial trehalose.
Statistical analysis
The experiment was carried out according to a split plot experiment based on a completely randomized design with three replications. Data were analyzed by PROC ANOVA procedure by SAS software (Ver. 9.1 2002–2003, SAS Institute, Cary, NC, USA). Before analysis of variance, data were tested for normality and homoscedasticity using the Kolmogorov–Smirnov and Cochran tests, respectively. Least significant difference (LSD) at P ≤ 0.01 and P ≤ 0.05 was calculated to compare differences between means following a significant ANOVA result.
Results and discussion Total fresh and dry weights
The results showed that after one week treatment, salinity stress significantly enhanced total FW in 10 dS m−1 of Hashemi parental genotype, but reduced total FW in other treatments (Table 1). Hashemi advanced mutant line 6 h after salinity stress showed the highest total DW in both treated and untreated seedlings as compared with Hashemi parental genotype. At the end of experiment, the highest total DW slightly increased in 10 dS m−1 of Hashemi parental genotype. Total DW of Hashemi advanced mutant after 1 w treatment significantly reduced by salinity stress, as this inhibitory effect increased with increasing concentrations of salinity (Table 1).
422 Table 1. Effect of salinity stress on seedling total fresh weight (FW) and dry weight (DW) in Hashemi parental and Hashemi advanced mutant line rice under Hydroponic conditions.
Sampling times Salinity Cultivar Total FW
(mg)
Total DW
(mg)
6 h 0 Parental 58.6 c 19.3 b
5 86.5 b 17.5 bc
10 64.0 c 16.5 c
0 Mutant 80.9 b 24.1 a
5 115.0 a 24.0 a
10 115.0 a 22.0 a
48 h 0 Parental 86.0 ab 23.0 b
5 77.5 b 22.0 b
10 99.5 a 29.5 a
0 Mutant 92.0 ab 24.5 ab
5 83.5 ab 26.0 ab
10 87.0 ab 24.5 ab
1 w 0 Parental 170.0 ab 39.0 a
5 100.0 d 24.5 b
10 175.0 a 39.5 a
0 Mutant 145.0 abc 39.0 a
5 135.0 bcd 32.5 ab
10 130.0 cd 30.0 ab
* For each column and sampling time, means followed by the same letters are not significantly different at P ≤ 0.01 according to the least significant difference test.
Salinity-induced fresh weight reduction is a common phenomenon for most of the cultivated crop plants and trees.
The reductions of fresh weight due to salinity stress have also been investigated by several scientists in several crops, whereas the increase in fresh weight in salinity has been reported by Mane et al. (2011). The reduction in biomass increased with the increase in salinity which is obvious because of disturbances in physiological and biochemical activities under saline conditions that may be due to the reduction in leaf area and number of leaves (Yunwei et al., 2007).
Moreover, the inhibitory effect of salinity stress on plant growth as well as plant fresh and dry weights could be due to the negative effect of salinity on the rate of photosynthesis, the changes in enzyme activity (that subsequently affects protein synthesis), and also the decrease in the level of carbohydrates and growth hormones, both of which can lead to inhibition of the plant growth and development (Mazher et al., 2007).
Total chlorophyll
As figure 1 shows, total chlorophyll content significantly decreased by increasing salinity stress severity, as the control treatment had the highest total chlorophyll content. Furthermore, Hashemi advanced mutant line slightly delayed the decreasing of total chlorophyll content and had the highest total chlorophyll content as compared with Hashemi parental genotype (Figure 1).
423 Figure 1. Change in total chlorophyll content of Hashemi parental and Hashemi advanced mutant line rice at different sampling times in response to salinity stress. For each sampling time, means followed by the same letters are not significantly different at P ≤ 0.01 according to the least significant difference test.
Our results are in agreement with Ali et al. (2004) who indicated that salinity stress significantly reduced chlorophylls content of different rice genotypes. Reduction in chlorophyll concentrations is probably due to the inhibitory effect of the accumulated Na+ and Cl- ions on the biosynthesis of the different chlorophyll fractions (Ali et al., 2004). Rapid and large accumulation of reactive oxygen species such as hydrogen peroxide is associated with degradation of chlorophyll (Farouk, 2011).
Chlorophylls content have been suggested as one of the parameters of salt tolerance in crop plants. In the salt tolerant genotypes (Hashemi advanced mutant line), less decreasing of chlorophyll content may be due to the high antioxidant enzyme activities in response to salt stress that prevented degradation of leaf chlorophyll content (Sairam and Srivastava, 2002). Furthermore, reduction of leaf's chlorophyll content maybe due to increase in activity of chlorophyll destroying enzyme that would lead to destruction in chlorosplast and instability of protein complexity of pigments (Movafegh et al., 2012).
Proline
Proline content of both genotypes significantly increased in response to salinity stress, as the proline content was found in 10 dS m−1 treatment (Figure 2). With increasing sampling time, proline content slightly increased in both genotypes. It was revealed that Hashemi advanced mutant line had the higher proline content as compared with Hashemi parental genotype (Figure 2).
424 Figure 2. Change in proline content of Hashemi parental and Hashemi advanced mutant line rice at different sampling times in response to salinity stress. For each sampling time, means followed by the same letters are not significantly different at P ≤ 0.01 according to the least significant difference test.
Proline is an important component of salt-stress responses of plants. The levels of proline in the plants is mainly designates their ability to tolerate or to adapt to saline conditions, since proline can work as an enzyme protectant under stress (Khedr et al., 2003). Accumulation of proline in plants tissues as a response to salt stress has been attributed to enzyme stabilization and/or osmoregulation system of the plant itself (Turan et al., 2007).
Proline accumulation is regulated by multiple factors, such as its synthesis, catabolism, utilization for protein synthesis and transport from other tissues. Proline accumulation in environmental tension such as saltiness could be due to denovo synthesis which reason of proline biosynthesis in salty stress is because of increase in level of 1- D-Piroline-5-carboxylate synthesis enzyme (Grewal, 2010). Additionally, Ueda et al (2007) demonstrated that proline transporter (HvProT) was highly expressed in the apical region of barley roots under salt stress.
Trehalose
It was found that salinity stress significantly enhanced trehalose content in both genotypes and trehalose content increased with increasing salinity stress severity (Figure 3). Moreover, 1 w after salinity treatment, Hashemi advanced mutant line showed the highest trehalose content than Hashemi parental genotypes (Figure 3).
425 Figure 3. Change in trehalose content of Hashemi parental and Hashemi advanced mutant line rice at different sampling times in response to salinity stress. For each sampling time, means followed by the same letters are not significantly different at P ≤ 0.01 according to the least significant difference test.
Trehalose can serve as a carbohydrate storage molecule as well as a transport sugar, similar to the function of sucrose. It can also stabilize proteins and membranes of plants when exposed to stress by replacing hydrogen bonding through polar residues, preventing protein denaturation and fusion of membranes (Iturriaga et al. 2009;
Redillas et al., 2012). However, trehalose production in dicot plants has resulted in morphological growth defects or altered metabolism (Suarez et al., 2009). Additionally, trehalose acts as a source of carbon and energy and as a protector against stresses (Iturriaga et al. 2009).
Garg et al. (2002) reported that the increased trehalose accumulation in rice correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stressed and unstressed conditions.
Moreover, overexpression of the trehalose-6-phosphate synthase gene OsTPS1 in transgenic rice revealed an increased tolerance to abiotic stresses compared to control plants (Li et al., 2011).
Conclusion
The results showed that total fresh and dry weights as well as total chlorophyll content significantly decreased in response to salinity stress, but proline and trehalose content significantly increased. The inhibitory effects of salinity stress significantly increased with increasing of its severity. As regards that Hashemi advanced mutant line had the highest total chlorophyll and osmoprotectants content; it can be conclude that Hashemi advanced mutant line is the salt-tolerant genotype.
Acknowledgment
The authors are grateful to the Department of Plant Breeding and Biotechnology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran, for financial support.
REFERENCES
Ali Y, Aslam Z, Ashraf MY, Tahir GR. 2004. Effect of salinity on chlorophyll concentration, leaf area, yield and yield components of rice genotypes grown under saline environment. Int J Environ Sci Technol 1(3):221–225.
Bates LS, Waldren RP, and Teare LD. 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207.
Djilianov D, Georgieva T, Moyankova D, Atanassov A, Shinozaki K, Smeeken SCM, 2005. Improved abiotic stress tolerance in plant by accumulation of osmoprotectants gene transfer approach. Biotechnol Biotechnol Eq 19:63–71.
Farouk S. 2011. Ascorbic Acid and α-Tocopherol Minimize Salt-Induced Wheat Leaf Senescence. J Stress Physiol Biochem 7(3):58–79.
426
Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ. 2002. Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci USA 99:15898–15903.
Grewal HS. 2010. Water uptake, water use efficiency, plant growth and ionic balance of wheat, barely,canola and chickpea plants on a sodic vertosol with variable subsoil NaCl salinity. Agric Water Manage 97:148–156.
Hashemi A, Gharechahi J, Nematzadeh G, Shekari F, Hosseini SA, Hosseini Salekdeh G. 2016. Two-dimensional blue native/SDS-PAGE analysis of whole cell lysateprotein complexes of rice in response to salt stress. J Plant Physiol 200:90–101.
Holm G. 1954. Chlorophyll mutations in barley. Acta Agric Scand 4:457–461.
Hossain MR, Pritchard J, Ford-Lloyd BV. 2016. Qualitative and quantitative variation in the mechanisms of salinity tolerance determined by multivariate assessment of diverse rice (Oryza sativa L.) genotypes. Plant Genetic Resour 14(2):91–100.
Hosseini S, Gharechahi J, Heidari M, Koobaz P, Abdollahi S, Mirzaei M, Nakhoda B. Hosseini Salekdeh G. 2015. Comparative proteomic and physiological characterization of two closely related rice genotypes with contrasting responses to salt stress. Funct Plant Biol 42:527–
542.
Iturriaga G, Suarez R, Nova-Franco B. 2009. Trehalose metabolism: from osmoprotection to signalling. Int J Mol Sci 10:3793–3810.
Khedr AHA, Abbas MA, Abdel Wahid AA, Quick WP, Abogadallah GM. 2003. Proline induces the expression of salt-stress-responsive proteins and may improve the adaptation of Pancratium maritimum L. to salt-stress. J Exp Bot 54(392):2553–2562.
Li HW, Zang BS, Deng XW, Wang XP. 2011. Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta 234:1007–1018.
Lutts S, Kinet JM, Bouharmont J. 1996. NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Ann Bot 78:389–98.
Mane AV, Karadge BA, Samant J.S. 2011. Salt stress induced alteration in growth characteristics of a grass Pennisetum alopecuroides. J Environ Biol 32(6):753–758.
Mazher AMA, El-Quesni EMF, Farahat MM. 2007. Responses of ornamental and woody trees to salinity. World J Agric Sci 3:386–395.
Mostofa MG, Hossain MA, Fujita M, Tran LP. 2015. Physiological and biochemical mechanisms associated with trehalose-induced copper- stress tolerance in rice. Sci Rep 5:11433. pp. 1–16.
Movafegh S, Razeghi Jadid R, Kiabi S. 2012. Effect of salinity stress on chlorophyll content, proline, water soluble carbohydrate, germination, growth and dry weight of three seedling barley (Hordeum vulgare L.) cultivars. J Stress Physiol Biochem 8:157–168.
Munns R, Tester M. 2008. Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681.
Munns R. 2002. Comparative physiology of salt and water stress. Plant Cell Environ 25:239–250.
Nounjan N, Nghia PT, Theerakulpisut P. 2012. Exogenous proline and trehalose promote recovery of rice seedlings from salt-stress and differentially modulate antioxidant enzymes and expression of related genes. J Plant Physiol 169:596–604.
Paul M. 2007. Trehalose 6-phosphate. Curr Opin Plant Biol 10:303–309.
Pires IS, Negrão S, Oliveira MM, Purugganan MD. 2015. Comprehensive phenotypic analysis of rice (Oryza sativa) response to salinity stress.
Physiol Plant 155:43–54.
Porra RJ, Thompson WA, Kriedmann PE 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents:vertification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochem Biophys Acta 975:384–394.
Qadir M, Quillérou E, Nangia V, Murtaza G, Singh M, Thomas RJ, Drechsel P, Noble AD. 2014. Economics of salt-induced land degradation and restoration. Nat Resour Forum 38:282–295.
Redillas MCFR, Park SH, Lee JW, Kim YS, Jeong JS, Jung H, 2012. Accumulation of trehalose increases soluble sugar contents in rice plants conferring tolerance to drought and salt stress. Plant Biotechnol Rep 6:89–96.
Sairam RK, Srivastava GC. 2002. Changes in antioxidant activity in sub-cellular fractions of tolerant and susceptible wheat genotypes in response to long term salt stress. Plant Sci 162:897–904.
Su J, Wu R. 2004. Stress-inducible synthesis of proline in transgenic rice confers faster growth under stress conditions than that with constitutive synthesis. Plant Sci 166:941–948.
Suarez R, Calderon C, Iturriaga G. 2009. Improved tolerance to multiple abiotic stresses in transgenic alfalfa accumulating trehalose. Crop Sci 49:1791–1799
Tripathy MK, Tiwari BS, Reddy MK, Deswal R, Sopory SK. 2015. Ectopic expression of PgRab7 in rice plants (Oryza sativa L.) results in differential tolerance at the vegetative and seed setting stage during salinity and drought stress. Protoplasma 1–16.
http://dx.doi.org/10.1007/s00709-015-0914-2
Tu Y, Jiang A, Gan L, Hossain M, Zhang J, Peng B, Xiong Y, Song Z, Cai D, Xu W, Zhang J, He Y. 2014. Genome duplication improves rice root resistance to salt stress, Rice 7:15. http://dx.doi.org/10.1186/s12284-014-0015-4
Turan MA, Kalkat V, Taban S. 2007. Salinity-induced stomatal resistance, proline, chlorophyll and Ion concentrations of bean. Int J Agric Res 2:
483–488.
Ueda A, Yamamoto-Yamane Y, Takabe T. 2007. Salt stress enhances proline utilization in the apical region of barley roots. Biochem Biophys Res Commun 355:61–66.
Yunwei D, Tingting J, Shuanglin D. 2007. Stress responses to rapid temperature changes of the juvenile sea cucumber (Apostichopus japonicus Selenka),” J Ocean University China 6(3):275–280.
