Differential Proteomics Analysis For The Leaf Intercellular Fluid From Cotton Under Osmotic Stress

Osmotic stress caused by the external factors such as drought, salinity and extreme temperature is one of the abiotic stresses that affects on plants’growth and development greatly, and seriously affects the production of cotton. The intercellular fluid between plant cells contains many physiological active molecules, which play a very important function role in plants’growth, development and environmental response and so on. But the intercellular fluid is vulnerable to be polluted by the cytoplasm constituents in the extraction process, related components especially proteins’content is extremely low and its’ separation and preparation is difficult, which limit the further research of the intercellular fluid between. By far, the related researches about the component of plant intercellular fluid especially proteins response to osmotic stress is very few.In this study, we improved the previous relevant methods firstly, and established suitable methods for the preparation of cotton leaf intercellular fluid and the extraction of intercellular fluid proteins; used the Mannitol to simulate the osmotic stress environment to investigate and analyze the effects of osmotic stress on morphological indexes of cotton seedlings and the activity expression of related enzymes in the antioxidant enzyme system in the cotton leaf intercellular fluid; then on this basis, we carried out the differential proteomics analysis for the leaf intercellular fluid from cotton under osmotic stress. The concrete research results are as follows:1. The extraction method of plant intercellular fluid was improved. Based on the previous penetration-centrifugation extraction method, more suitable penetrants and the penetrating method was selected in the study. Then the intercellular fluid was obtained by centrifugation. This method is not only simple but the cytoplasmic contamination of cotton intercellular fluid is lower which increases the reliability of the test materials.2. Due to the less intercellular fluid protein content, a method which is suitable for precipitating proteins from dilute aqueous solution was established. The intercellular fluid proteins couldn’t be extracted by traditional trichloroacetic acid-acetone method very well. So we used the lauric acid-assisted trichloroacetic acid-acetone precipitation method in the study. Results indicated that Lauric acid as a carrier of protein precipitation could preferably precipitate the intercellular fluid proteins and adapte to the subsequent test.3. The effects of osmotic stress on morphological indexes of cotton seedlings. Under conditions of stress treatment for the same time, the treatment group cotton leaves were varying degrees of dehydration as osmotic stress level increased, and the degree of wilting leaves of cotton compared to the control more serious when concentration was 600 mM; Under conditions of stress treatment at the same level, the cotton leaves were varying degrees of dehydration as the treatment time from 0 h to 72 h, and the degree of cotton leaves wilted more serious when treatment time was 72 h;4. The expression of antioxidant enzymes system (Superoxide dismutase, SOD; Peroxidase, POD; Catalase, CAT; Ascorbate peroxidase, APOX) in the cotton leaf intercellular fluid under osmotic stress was measured, the analysis indicated that osmotic stress could affect the expression of antioxidant enzymes system in the cotton leaf intercellular fluid. The expression of POD in cotton intercellular fluid had varying degrees of decline under osmotic stress for from 0 h to 60 h, and the activity of POD showed the highest decline at 60 h during treat course which is 0.078 times as is control. The expression of CAT and APOX in cotton intercellular fluid promoted under osmotic stress for from 0 h to 60 h, and the activity of CAT and APOX was respectively achieved the highest folds of 1.2 and 2.33 to control at 60 h.5. The proteins of cotton leaf intercellular fluid under osmotic stress were separated by the Two-dimensional electrophoresis which is based on the O’Farrell’s method, and obtained the protein gel images with clear and consistent background. The gel images were analyzed by PD Quest 8.0.1 software package. Results showed that the expression of 9 proteins were up-regulated while 4 proteins were down-regulated under osmotic stress.6. These proteins were analyzed by tandem mass spectrometry (MS/MS). The identified proteins includes beta-1,3-glucanase, Serine carboxypeptidase-like 51, desiccation-related pcC 13-62, chitinase class IV and plant basic secretory protein (BSP) family protein which were related to osmotic stress. The modified methods of collecting intercellular fluid and extracting proteins from diluted solutions in this paper will facilitate the studies of the leaf intercellular fluid. The homologous identification of the cotton leaf intercellular fluid proteins provide certain theoretical basis for finding the cotton osmotic resistance mechanism.