بررسی الگوی الکتروفورز دو بعدی ریشه‌ی کلزای تلقیح شده با باکتری Pseudomonas fluorescens FY32 تحت تنش شوری

نوع مقاله : علمی - پژوهشی

نویسندگان

1 دانش‌آموخته کارشناسی ‌ارشد، گروه بهنژادی و بیوتکنولوژی، دانشکده کشاورزی، دانشگاه تبریز، تبریز

2 دانشیار، گروه بهنژادی و بیوتکنولوژی، دانشکده کشاورزی، دانشگاه تبریز، تبریز

3 دانشیار، گروه زیست‌شناسی، دانشکده علوم پایه، دانشگاه شهید مدنی آذربایجان، تبریز

چکیده

کلزا که به‌منظور استحصال روغن آن تولید می‌گردد، همانند سایر گیاهان زراعی متأثر از شوری می‌شود. شوری خاک به‌عنوان یکی از مهمترین عوامل نامساعد و تنش‌زای غیرزنده اثر نامطلوب بر عملکرد و کیفیت محصولات کشاورزی دارد. باکتریPseudomonasfluorescens جزو باکتری‌های محرک رشد گیاه می‌باشد و در همیاری با گیاهان باعث افزایش رشد مقاومت گیاه در برابر عوامل بیماری‌زا و محدود‌کننده محیطی می­گردد. در این تحقیق شوری در دو سطح صفر و 300 میلی‌مولار نمک NaCl اعمال شد. حضور یا عدم حضور باکتری به‌عنوان تیمارهای دیگر با سه تکرار اعمال گردید و برای تجزیه‌های آماری از طرح کرت‌های خرد شده با طرح پایه‌‌ی کاملاً تصادفی استفاده شد. به‌منظور بررسی پروتئین‌های دخیل در بهبود مقاومت گیاه کلزا به شوری در همیاری با باکتری Pseudomonas fluorescens الگوی الکتروفورز دو بعدی پروتئوم ریشه آن مورد بررسی قرار گرفت. نتایج حاصل نشان داد که از 216 لکه‌ی ظاهر شده 16 لکه‌ی پروتئینی در اثر اعمال تنش شوری، 13 لکه‌ی پروتئینی در حضور باکتری محرک رشد گیاه و 23 لکه در صورت اعمال هر دو تیمار تغییر بیان نشان می‌دهند. پس از شناسایی احتمالی پروتئین‌های مربوطه با استفاده از معیارهای وزن مولکولی و نقطه‌ی ایزوالکتریک مشخص شد این پروتئین‌ها که با افزایش یا کاهش بیان همراه بوده‌اند جزو پروتئین‌های دخیل در مسیرهای متابولیکی/ انرژی‌زایی، پیام‌رسانی، حفاظت و دفاع سلولی، کانالی، ساختار و مکان‌یابی پروتئین‌های دیگر، ساختمان سلولی و رونویسی و ترجمه هستند. با اعمال تیمار شوری یا باکتری یا شوری به‌همراه باکتری، بیشترین تعداد لکه‌های تغییر بیان یافته مربوط به گروه پروتئین‌های متابولیکی/ انرژی‌زایی بود.

کلیدواژه‌ها


عنوان مقاله [English]

Proteome Analysis of Canola Root Inoculated with Pseudomonas fluorescens FY32 under Salinity Stress

نویسندگان [English]

  • Pouya Motie Noparvar 1
  • Ali Bandehagh 2
  • Davood Farajzadeh 3
  • Ebrahim Dorani Uliaie 2
1 M.Sc. Graduated, Department of Plant Biotechnology and Breeding, Faculty of Agriculture, University of Tabriz, Tabriz
2 Associate Professor, Department of Plant Biotechnology and Breeding, Faculty of Agriculture, University of Tabriz, Tabriz
3 Associate Professor, Department of Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz
چکیده [English]

Canola which is being used for its oil is affected by salinity stress. Salinity is one of the most important abiotic stressful conditions which affects productivity and quality of agricultural products. Pseudomonas fluorescens is a Plant growth promoting rhizobacteria having symbiotic relation with plants that reduces adverse effects and increases plant growth. In this study salinity stress has been applied in two levels (0 and 300 mM NaCl). Absence and presence of P. fluorescens was the other treatment applied to plant root with three replications. Statistical analysis was carried out as split plot based on completely randomized design. In order to evaluate proteome analysis of canola root under salinity stress inoculated with P. fluorescens 2D-PAGE approach was carried out. To reach this goal proteome extract of root was extracted via TCA-acetone method. Isoelectric focusing and SDS-PAGE for 1st and 2nd dimensions, were used, respectively. Acquired gels were analyzed using PD-quest software after staining with silver nitrate. Results demonstrated 216 spots on the gels. Sixteen spots showed significant change as result of salinity stress. Thirteen spots showed change in quantity as result of presence of P. fluorescens FY32. After applying salinity and inoculation PGPR, 23 spots presented changes significantly. After identification of proteins using molecular weight and PI, it has been recognized that most of the responding proteins to salinity and PGPR presence conditions are categorized in metabolic/ energy related pathways. Then, signaling, maintenance/ defense, channel proteins and protein structure/ location related proteins were the ones that had high frequency respectively.

کلیدواژه‌ها [English]

  • 2D-PAGE
  • PGPR
  • Proteome analysis
  • root
  • Canola
Andème Ondzighi, C., Christopher, D. A., Cho, E. J., Chang, S. C. and Staehelin, L. A. 2008. Arabidopsis protein disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds. Plant Cell, 20: 2205-2220.
Apse, M. P. and Blumwald, E. 2007. Na+ transport in plants. FEBS Letters, 581: 2247-2254.
Arshad, M., William, JR. and Frankenberger, T. 2002. The Plant Hormone, Ethylene. Springer, pp. 342.
Bandeh-hagh, A., Toorchi, M., Mohammadi, A., Chaparzadeh, N., Hosseini-Salekdeh, G. H. and Kazemnia, H. 2008. Growth and osmotic adjustment of canola genotypes in response to salinity. Journal of Food, Agriculture and Environment, 6: 201-208.
Bauwe, H. and Kolukisaoglu, U. 2003. Genetic manipulation of glycine decarboxylation. Journal of Experimental Botany, 54: 1523-1535.
Bazyar, M. 2013. Effects of salinity stress on activity of some antioxidant enzymes and root characteristics of canola cultivars inoculated with Pseudomonas fluorescens FY32 (M. S. Dissertation). Faculty of Agriculture, University of Tabriz. pp 102.
Berth, M., Moser, F. M., Kolbe, M. and Bernhardt, J. 2007. The state of the art in the analysis of two-dimensional gel electrophoresis images. Applied Microbiology and Biotechnology, 76(6): 1223-1243.
Bowler, C., Van Montagu, M. and Inze, D. 1992. Superoxide dismutase and stress tolerance. Annual Review of Plant Physiology and Plant Molecular Biology, 43: 83-116.
Bradford, M. M. 1976. A dye binding assay for protein. Analytical Biochemistry, 72: 248-254.
Cai, H., Wang, C. C. and Tsou, C. L. 1994. Chaperone-like activity of protein disulfide isomerase in the refolding of a protein with no disulfide bonds. The Journal of Biological Chemistry, 269: 24550-24552.
Cárdenas, L., Vidali, L., Domínguez, J., Pérez, H. and Sánchez, F. 1998. Rearrangement of actin microfilaments in plant root hairs responding to Rhizobium etli nodulation signals. Plant Physiology, 116: 871-877.
Cheng, Y., Qi, Y., Zhu, Q., Chen, X., Wang, N. and Zhao, X. 2009. New changes in the plasma membrane associated proteome of rice roots under salt stress. Proteomics, 9: 3100-3114.
Dangl, J. L., Preuss, D. and Schroeder, J. I. 1995. Talking through walls: Signaling in plant development. The Journal Cell, 83: 1071-1077.
De la Fuente, J. M., Ramirez-Rodrigez, V., Cabrera-Ponce, J. S. and Herrera-Esterella, L. 1997. Aluminum tolerance in transgenic plants by alteration in citrate synthesis. Science, 276: 1566-1568.
Domash, V. I., Sharpio, T. P., Zabreĭko, S. A. and Sosnovskaia, T. F. 2008. Proteolytic enzymes and trypsin inhibitors of higher plants under stress conditions. Bioorganicheskaia Khimiia, 34: 353-357.
Dubey, R. S. 1997. Photosynthesis in plants under stressful conditions. In: Pessarakli, M. (ed.).Handbook of plant and crop stress. New York: Marcel Dekker, Incorporation. pp: 859-875.
Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A. and Rutter, W. J. 1985. Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. Nature, 317: 267-270.
Farajzadeh, D., Aliasgharzad, N., Sokhandan Bashir, N. and Yakhchali, B. 2010. Cloning and characterization of a plasmid encoded ACC deaminase from an indigenous (Pseudomonas sp. FY32). Current Microbiology Journal, 61: 37-43.
Francois, L. E. 1994. Growth, seed yield and oil content of canola grown under saline conditions. Agron. J., 86: 233–237.
Francois, L. E. and Maas, E. V. 1994. Crop response and management on salt-affected soils. In: Pessarakli, M. (ed.).Handbook of plant and crop stress. New York: Marcel Dekker, Incorporation. pp: 149-181.
Fujita, Y., Fujita, M., Shinozaki, K. and Yamaguchi-Shinozaki, K. 2011. ABA-mediated transcriptional regulation in response to osmotic stress in plants. Journal of Plant Research, 124: 509-525.
Ghosh, D. and Xu, J. 2014. Abiotic stress responses in plant roots: A proteomics perspective. Frontiers in Plant Science, 5(6): 1-13.
Haas, D. and Keel, C. 2003. Regulation of antibiotic production in root-colonizing Pseudomonas spp. and relevance for biological control of plant disease. Annual Review of Phytopathology, 41: 117-153.
Henty-Ridilla, J. L., Shimono, M., Li, J., Chang, J. H., Day, B. and Staiger, C. J. 2013. The plant actin cytoskeleton responds to signals from microbe-associated molecular patterns. Journal of Pathogens, 9: 1-14.
Horie, T. and Schroeder, J. I. 2004. Sodium transporters in plants. Diverse genes and physiological functions. Plant Physiology, 136: 2457-2462.
Kim, Y. H., Kim, M. D., Choi, Y. I., Park, S. C., Yun, D. J., Noh, E. W., Lee, H. S. and Kwak, S. S. 2011. Transgenic poplar expressing Arabidopsis NDPK2 enhances growth as well as oxidative stress tolerance. Plant Biotechnology Journal, 9: 334-347.
Klappa, P., Ruddock, L. W., Darby, N. J. and Freedman, R. B. 1998. The b′ domain provides the principal peptide-binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins. European Molecular Biology Organization journal, 17: 927-935.
Kloepper, J. W. 1993. Plant growth-promoting rhizobacteria as biological control agents. In: Metting, F. B. (ed.). Soil Microbial Ecology: Applications in Agricultural and Environmental Management. Marcel Dekker, Incorporation. pp. 255-274.
Koyama, H., Kawamura, A., Kihara, T., Hara, T., Takita, E. and Shibata, D. 2000. Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus-limited soil. Plant Cell Physiology, 41: 1030-1037.
Koyama, H., Ojima, K. and Yamaya, T. 1992. Characteristics of aluminum-phosphate-adapted carrot cells: uptake and utilization of phosphate. Plant Cell Physiology, 33: 171-176.
Lee, R. H., Lin, M. C. and Chen, S. C. 2004. A novel alkaline alpha-galactosidase gene is involved in rice leaf senescence. Plant Molecular Biology, 55: 281-295.
Leterrier, M., Del Río, L. A. and Corpas, F. J. 2007. Cytosolic NADP-isocitrate dehydrogenase of pea plants: genomic clone characterization and functional analysis under abiotic stress conditions. Free Radical Research, 41: 191-199.
Maurel, C., Santoni, V., Luu, D. T., Wudick, M. M. and Verdoucq, L. 2009. The cellular dynamics of plant aquaporin expression and functions. Current Opinions in Plant Biology, 12: 690-698.
McFarland, J. 1907. The nephelometer: an instrument for estimating the numbers of bacteria in suspensions used for calculating the opsonic index and for vaccines. The Journal of the American Medical Association, 49: 1176-1178.
Moons, A., Gielen, J., Vandekerckhove, J., Van Der Straeten, D., Gheysen, G. and Van Montagu, M. 1997. An abscisic acid and salt stress responsive rice cDNA from a novel plant gene family. Planta, 202: 443-454.
Motie Noparvar, P. 2014. Proteome analysis of canola root inoculated with Pseudomonas fluorescens FY32 under salinity stress (M.S. Dissertation). Faculty of agriculture, University of Tabriz. PP 98.
Munns, R. and Termaat, A. 1986. Whole-plant responses to salinity. Australian Journal of Plant Physiology, 13: 143-160.
Onda, Y., Nagamine, A., Sakurai, M., Kumamaru, T. and Ogawa, M. 2011. Distinct roles of protein disulfide isomerase and P5 sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice. Plant Cell, 23: 210-223.
Palleroni, N. J. 1984. Pseudomonadaceae. Bergey's Manual of Systematic Bacteriology. In: Krieg, N. R. and Holt, J. G. (eds.). Baltimore: The Williams and Wilkins Co., pp. 141-199.
Ricard, B., Rivoal, J., Spiteri, A. and Pradet, A. 1991. Anaerobic stress induces the transcription and translation of sucrose synthase in rice. Plant Physiology, 95: 669-674.
Rodriguez, P. L. 1998. Protein phosphatase 2C (PP2C) function in higher plants. Plant Molecular Biology, 38: 919-927.
Romero-Aranda, R., Soria, T. and Cuartero, J. 2001. Tomato plant-water uptake and plant-water relationships under saline growth conditions. Plant Science, 160: 265-272.
Sánchez, A., Rodríguez, G. J. M., García R., Torreblanca, J. and Pardo, J. M. 2004. Salt stress enhances xylem development and expression of S-adenosyl-L-methionine synthase in lignifying tissues of tomato plants. Planta, 220: 278-285.
Schaffner, A. R. 1998. Aquaporin function, structure, and expression: are there more surprises to surface in water relations?, Planta, 204: 131-39.
Schulz, P., Herde, M. and Romeis, T. 2013. Calcium-dependent protein kinases: Hubs in plant stress signaling and development. Plant Physiology, 163: 523-530.
Shannon, M. C. and Grieve, C. M. 1999. Tolerance of vegetable crops to salinity. Scientia Horticulturae, 78: 5-38.
Smertenko, A. and Franklin-Tong, V. E. 2011. Organisation and regulation of the cytoskeleton in plant programmed cell death. Cell Death and Differentiation, 18: 1263-1270.
Staiger, C. J., Sheahan, M. B., Khurana, P., Wang, X. and McCurdy, D. W. 2009. Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array. Journal of Cell Biology, 184: 269-280.
Strange, R. C., Spiteri, M. A., Ramachandran, S. and Fryer, A. A. 2001. Glutathione-S-transferase family of enzymes. Mutation Research, 482: 21-26.
Tsai, B., Rodighiero, C., Lencer, W. I. and Rapoport, T. A. 2001. Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin. The Journal Cell, 104: 937-948.
Tyerman, S. D., Bohnert, H. J., Maurel, C., Steudle, E. and Smith, J. A. 1999. Plant aquaporins: their molecular biology, biophysics and significance for plant water relations. Journal of Experimental Botany, 50: 1055-1071.
Waditee, R., Bhuiyan, N. H., Hirata, E., Hibino, T., Tanaka, Y., Shikata, M. and Takabe, T. 2007. Metabolic engineering for betaine accumulation in microbes and plants. Journal of Biological Chemistry, 282: 34185-34193.
Wilkinson, B. and Gilbert, H. F. 2004. Protein disulfide isomerase. Biochimica et Biophysica Acta, 1699: 35-44.
Xu, Y., Zhan, C. and Huang, B. 2011. Heat shock proteins in association with heat tolerance in grasses. Inter. Journal of Proteomics, 2011: 1-11.
Yeo, A. 1998. Molecular biology of salt tolerance in the context of whole-plant physiology. Journal of Experimental Botany, 49: 913-929.