Comparative proteomic analysis of seedling leaves of different salt tolerant soybean genotypes
Graphical abstract
Highlights
► 78 proteins were identified by 2-DE coupled with MS/MS in soybean seedling leaves. ► ROS and ethylene signal transduction pathways were enhanced by salt stress. ► Synthesis and emission of ethylene were positively correlated with salt tolerance. ► Energy depletion was increased in soybean seedling leaves in response to salt stress. ► A possible salt stress-responsive protein network is proposed.
Introduction
Salinity is one of the most widespread agricultural problems in arid and semi-arid regions around the world that make fields unproductive [1]. It is estimated that about 20% of irrigated land, which yields one-third of the world's food, is affected by salinity [2]. Moreover, a significant proportion of recently cultivated agricultural land has become saline because of land clearing or irrigation [3]. As a result, the development of improved levels of tolerance to salt stress has become an urgent priority for many crop breeding programs [4], and much research effort has been applied to gain a better understanding of the adaptive mechanisms adopted by plants to combat salt stress.
Soybean is an important dicot crop due to the high content of oil and protein in its seeds [5]. More than one-third of the world's edible oils and two-thirds of the world's protein meal are derived from soybean [6]. However, soybean is subject to salinity stress that reduces its yield like many other crops. Therefore, the demand for developing salt-tolerant soybean cultivars is unavoidable. Two genotypes, Jackson and Lee 68, have been widely used to reveal soybean responses to salinity stress at physiological and agronomic trait levels by many investigators [7], [8]. Pantalone et al. reported that genotype Jackson accumulated more chloride in the leaves and had significantly higher leaf chlorosis score than genotype Lee 68 at the same salt level [9]. Hamwieh et al. reported that Jackson was a salt-sensitive soybean genotype and had significantly lower salt-tolerance ratings than genotype JWS156-1 [10]. Under 150 mM NaCl stress, Cl− was found to be more toxic than Na+ to soybean seedlings and injury of soybean genotypes, including ‘Jackson’ (salt sensitive) and ‘Lee 68’ (salt tolerant), was positively correlated with the content of Cl− in the leaves [8]. Valencia et al. established a rapid and effective method for screening salt tolerant soybean genotypes based on foliar symptoms with genotype Lee 68 used as one of the salt-tolerant genotypes [11]. Their results indicated that salt sensitive genotypes exhibited interveinal chlorosis while salt tolerant genotypes (including Lee 68) showed no chlorosis at the 120 mM NaCl level, and average leaf Cl− content for salt sensitive genotypes was 1.96 times higher than for salt tolerant genotypes (including Lee 68) [11]. Taken together, all above previous results suggested that Jackson was a salt sensitive genotype while Lee 68 was a salt tolerant genotype. But their molecular mechanism in response to salt stress still remains unclear.
Proteomics is a powerful molecular tool for describing the complete proteome at organelle or tissue level and for comparing how the proteome is affected by different physiological conditions [6]. Two-dimensional polyacrylamide gel electrophoresis (2-DE) is one of the most sensitive and powerful techniques for separating hundreds of proteins [12]. It has been applied to different abiotic treatments for soybean including treatment with salt and flooding, and exposure to ultraviolet radiation [13], [14]. Aghaei et al. identified seven proteins in soybean hypocotyl and root under salt stress and indicated that salinity can change the expression level of some special proteins that might play a role in the adaptation to saline conditions [15]. Sobhanian et al. identified salt responsive proteins in soybean using a proteomic technique and concluded that glyceraldehyde-3-phosphate dehydrogenase was down-regulated in the leaf/hypocotyls, and fructokinase 2 was down-regulated in the hypocotyls/root under NaCl treatment, while stem 31 kDa glycoprotein precursor was up-regulated in leaf/hypocotyls/root [6]. Xu et al. carried out a proteomic analysis of seed germination under salt stress in soybean and identified nine proteins by MALDI-TOF-MS [2]. Recently, Sobhanian et al. reviewed the changes in the plant proteome that resulted from salt stress and summarized the changes of metabolic pathways of soybean under salt stress. They indicated that photosynthesis, protein biosynthesis and ATP biosynthesis were decreased while defense protein increased in soybean in response to salt stress [16]. However, to date, still only limited information is available about salt-response proteins in soybean. This has limited our understanding of the molecular mechanism adopted by this important crop in response to salt stress.
In the present study, a proteomic approach was applied to seedling leaves of two soybean genotypes, Jackson (salt-sensitive) and Lee 68 (salt-tolerant). The main objectives were to investigate the proteome expression pattern and to identify the differentially expressed proteins under salt stress; and, based on the proteomic data, the molecular mechanism in responses to salinity stress in soybean seedling leaves was discussed. Additionally, why genotype Lee 68 is more salt tolerant than genotype Jackson was also discussed at molecular level.
Section snippets
Plant material
Seeds of two soybean genotypes (salt-sensitive genotype Jackson and salt-tolerant genotype Lee 68) [7], [8] were sterilized with 0.1% HgCl2 for 10 min. After three times of rinsing with sterilized distilled water, the seeds were germinated on wet filter paper in the dark for 72 h at 26 °C. Uniformly germinated seeds were transplanted into 1/2 Hoagland nutrient solution, which was replaced with fresh one every 3 days. The seedlings were grown in a growth chamber with 25/20 °C temperature (day/night),
2-DE analysis and identification and functional classification of differentially expressed proteins
In this study, 2-DE maps were obtained using IEF on 24 cm pH 4–7 nonlinear IPG gels followed by SDS-PAGE on 12% polyacrylamide gels. The 2-DE proteome profiles of each sample were stained by CCB G250 and the representative results are shown in Supplementary 1. The results revealed a consistent protein pattern in their expression levels on the gels. The 2-DE profile showed that approximate 800 protein spots could be reproducibly detected mainly in the range of pH 4–7 and relative molecular masses
Conclusions
To investigate changes of global proteins under salt stress, we performed a comparative proteome analysis of the seedling leaves of two soybean genotypes (salt tolerant genotype Lee 68 and salt sensitive genotype Jackson) using NaCl as a model for salt stress. 91 protein spots on the 2-DE gel image containing about 800 reproducible spots were found to be differentially expressed in salt-treated soybean seedling leaves. Of them, 78 were successfully identified by MALDI-TOF–TOF. These identified
Acknowledgments
We gratefully acknowledge the partial financial support from the projects supported by the National Natural Science Foundation of China (30971840, 31171572, 31101212), from the project supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (20100097110030), and from the Science and Technology Development Foundation of Shanghai Agricultural Academy (2000-04-06-3). We thank Prof. Bingjun Yu, from the College of Life Sciences, Nanjing Agricultural
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Contributed equally to this work.