بررسی الگوی بیان ژن‌های P5CS وCBF تحت تنش یخ‌زدگی و اثر خوسرمایی بر تغییرات میزان پرولین آزاد در گیاه نخود (Cicer arietinum L.)

نوع مقاله : مقالات پژوهشی

نویسندگان

• روح الله قاسمی ¹
• عبدالرضا باقری ²
• نسرین مشتاقی ²
• فرهاد شکوهی فر ³

¹ دانشگاه تربیت مدرس

² دانشگاه فردوسی مشهد

³ پژوهشکده علوم گیاهی دانشگاه فردوسی مشهد

10.22067/ijpr.v8i2.34624

چکیده

سازگاری گیاهان به تنش یخ‌زدگی از طریق تنظیم فعالیت ژن‌ها و متابولیسم سلولی انجام می‌شود. در این پژوهش، تغییرات بیان ژن‌های P5CS و (DREB1)CBF در شرایط دمایی معمولی (23‌درجه سانتی‌گراد)، خوسرمایی (10درجه سانتی‌گراد) و یخ‌زدگی (10-‌درجه سانتی‌گراد) در دو ژنوتیپ نخود زراعی مقاوم (MCC426) و حساس (MCC505) به سرما مورد ارزیابی قرار گرفت. نتایج آنالیز داده‌ها نشان داد که بیان این ژن‌ها در ژنوتیپ مقاوم در مقایسه با ژنوتیپ حساس به‌طور معنی‌داری بیشتر بود (P

کلیدواژه‌ها

• بیان ژن
• تنش یخ‌زدگی
• پرولین
• نخود زراعی

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عنوان مقاله [English]

The pattern of CBF and P5CS genes expression under freezing stress and effects of cold acclimation on free proline changes in chickpea (Cicer arietinum)

نویسندگان [English]

• ROHOLLAH GHASEMI ¹
• Abdolreza Bagheri ²
• Nasrin Moshtaghi ²
• Farhad Shokouhi Far ³

¹ Ferdowsi University of Mashhad

² Ferdowsi University of Mashhad

³ Ferdowsi University of Mashhad

چکیده [English]

Introduction
Cold and freezing stress is one of the most important environmental stresses that affect the growth and yield of crops. One of the important processes in plant resistance to cold stress is known to acclimatize. In this process, the internal mechanisms of plants causes higher resistance to freezing stress after exposure to temperatures close to zero. Low temperature stress induce or inhibit the expression of certain genes. P5CS protein and transcription factor DREB (CBF) play an important role in adaptation and cold tolerance in plants. P5CS activates the enzymes in proline synthesis pathway and cause accumulation of this substance in plant that protects membranes and proteins against different effects of high concentrations of inorganic ions and high and low temperatures. Also DREB proteins with a conserved sequence bind to specific DNA sequences upstream of responsible genes in resistance to cold stress and leads to their expression. In this study the expression of P5CS and (DREB1)CBF genes that have an important role in the face of many plants to freezing and cold stress were examined in chickpea in both resistant and susceptible genotypes and used of Real time PCR method to evaluate the expression of these genes under stress and non-stress.

Materials & Methods
In the first experiment, the expression pattern of P5CS and CBF (DREB1) genes were evaluated at normal (23°C), acclimation (10°C) and freezing (-10°C)temperatures in two resistant (MCC426) and susceptible (MCC505) genotypes of chickpea, in two organs (leaf and stem). Briefly pots containing plants of both genotypes stored at 23°C for two weeks. After this period, plants are divided into two parts, half remained in the normal conditions and the other half were transferred to 10°C for cold acclimation. After 10 days, seedlings of normal and acclimation conditions transferred to a thermogradient freezer and after 15 minutes at -10°C, sampling was carried out from the leaves and stems. The simultaneous sampling of the control plants (normal conditions) were also done. After RNA extracting from samples and determine the concentration and quality by nanodrop and treatment with DNase I enzyme, cDNA synthesis was performed. After designing appropriate primers, the PCR process was optimized and Real time PCR was performed under optimized process conditions and gene expression levels were calculated. In the Second experiment free proline changes of seedlings in the acclimation phase were evaluated. Briefly after transferring of plants in the acclimation conditions, amount of proline at them were measured at appropriate intervals after exposure in acclimation conditions.

Results & Discussion
Results of analysis showed that the expression of these genes were significantly higher in resistant genotype than the susceptible one. Because of these genes are the important factors in plant resistance to stress especially cold and freezing stress, this can confirm that MCC426 genotype is resistant and MCC505 genotype is sensitive to cold. Also the interaction of genotype and organ sample in the control condition showed significant difference only for expression of P5CS gene. Since this gene is an important gene in freezing and cold stress, increased expression of this gene in MCC426 genotype that is resistant to cold not unexpected. But expression of CBF gene occurred only after cold stress induction and in the absence of stress is poor expression. In addition, it was revealed that the interaction of genotype and acclimation treatment on CBF genes in both genotypes had significant differences, but in P5CS gene only in MCC426, this trend was observed. However, changes of free proline as one of the mechanisms of cell protection in acclimation period in both genotypes showed that proline contents of MCC426 in response to acclimation treatment was gradually increased, but this increase in MCC505 genotype occurred during the early stages of acclimation, and proline has decreased over time.

Conclusion
According to the results, it generally seems, one of the main reason for tolerance to chilling is the high expression levels of the mentioned genes during chilling and freezing stress and therefore it can be used for inducing freezing tolerance into sensitive plants.

کلیدواژه‌ها [English]

• Cicer arietinum
• Gene expression

مراجع

Agarwal, P.K., Agarwal, P., Reddy, M., and Sopory, S. K. 2006. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Reports 25: 1263-1274.

2. Bates, L.S. 1973. Rapid determination of free proline for water stress studies. Plant and Soil 39: 205-207.

3. Delauney, A., Hu, C., Kishor, P., and Verma, D. 1993. Cloning of ornithine delta-aminotransferase cDNA from Vignaaconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis. Journal of Biological Chemistry 268: 18673-18678.

4. Dibax, R., Deschamps, C., Bespalhok Filho, J., Vieira, L.G.E., Molinari, H.B.C., DeCampos, M.K.F., and Quoirin, M. 2010. Organogenesis and Agrobacterium tumefaciens-mediated transformation of Eucalyptus saligna with P5CS gene. Biologia Plantarum 54: 6-12.

5. Dinari, A., Niazi, A., Afsharifar, A. R., and Ramezani, A. 2013. Identification of upregulated genes under cold stress in cold-tolerant chickpea using the cDNA-AFLP approach. PloS one 8(1): e52757.

6. Dörffling, K., Schulenburg, S., Lesselich, G., and Dörffling, H. 2008. Abscisic acid and proline levels in cold hardened winter wheat leaves in relation to variety‐specific differences in freezing resistance. Journal of Agronomy and Crop Science 165: 230-239.

7. Esfandiari, E., Shekari, F., and Esfandiari, M. 2007. The effect of salt stress on antioxidant enzymes' activity and lipid peroxidation on the wheat seedling. Journal of Notulae Botanicae Horti Agribotanici Cluj-Napoca 35: 48-56.

8. Filippi, D.L., Fournier, M., Cameroni, E., Linder, P., Virgilio, C.D., Foti, M., and Deloche, O. 2007. Membrane stress is coupled to a rapid translational control of gene expression in chlorpromazine-treated cells. Current Genetics 52: 171-185.

9. Fowler, S., and Thomashow, M.F. 2002. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 14: 1675-90.

10. Gentle, A., Anastasopoulos, F., and Mc Brien, N. A. 2001. High resolution semi quantitative real time PCR without the use of a standard curve. BioTechniques 31: 502-508.

11. Gilmour, S.J., Sebolt, A.M., Salazar, M.P., Everard, J.D., and Thomashow, M.F.2000. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiology 124:1854-65.

12. Gilmour, S.J., Zarka, D.G., Stockinger, E.J., Salazar, M.P., Houghton, J.M., and Thomashow, M.F. 2001. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold‐induced COR gene expression. The Plant Journal 16: 433-442.

13. Gusta, L.V., Trischuk, R., and Weiser, C.J. 2007. Plant cold acclimation: The role of abscisic acid. Journal of Plant Growth Regulation 24: 308-318.

14. Harding, M.M., Ward, L.G., and Haymet, A.D.J. 1999.Type I `antifreeze' proteins: Structureactivity studies and mechanisms of ice growth inhibition. European Journal of Biochemistry 264: 653-665.

15. Hsieh, T.H., Lee, J.T., Yang, P.T., Chiu, L.H., Charng, Y., Wang, Y.C., and Chan, M.T. 2002. Heterology expression of the Arabidopsisc-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato. Plant Physiology 129: 1086-1094.

16. Jaglo, K.R., Kleff, S., Amundsen, K.L., Zhang, X., Haake, V., Zhang, J.Z., and Thomashow, M. F. 2001. Components of the Arabidopsisc-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in brassica napus and other plant species. Plant Physiology127: 910-917.

17. Kafi, M., and Damghani, A.M. 2000. Resistance Mechanisms of Plants to Environmental Stresses. Ferdowsi University of Mashhad Press.

18. Konstantinova, T., Parvanova, D., Atanassov, A., and Djilianov, D. 2002. Freezing tolerant tobacco, transformed to accumulate osmoprotectants. Plant Science 163: 157-164.

19. Murry, G.A., Eser, D. , Gusta, L.V., and Eteve, G. 1988. Winter hardiness in pea, lentil, faba bean and chickpea. In R.J. Summerfield (Ed.) World Crops: Cool Season Food Legumes. Kluwer Academic Publishers.The Netherlands. P. 831-843.

20. Nayyar, H., Bains, T.S., and Kumar, S. 2005. Chilling stressed chickpea seedling: effect of cold acclimation, calcium and abscisic acid on cryoprotective solutes and oxidative damage. Environmental and Experimental Botany 54: 275-285.

21. Nezami, A., and Bagheri, A. 2001. Screening of Mashhad chickpea (Cicer arietinum L.) collection for cold tolerance under field conditions. Agricultural Sciences and Technology 15:155-162.

22. Rayapati, P.J., and Stewart, C.R. 1991. Solubilization of a proline dehydrogenase from maize (Zea mays L.) mitochondria. Plant Physiology 95: 787-791.

23. Singh, K.B., Malhotra, R.S., and Saxena, M.C. 1995. Additional sources of tolerance to cold in cultivated and wild Cicer species. Crop Science 35: 1491-1497.

24. Thomashow, M.F. 1999. Plant cold acclimation:freezing tolerance genes and regulatory mechanisms. Annual Review of Plant Physiology and Plant Molecular Biology 50: 571-599.

25. Van swaaij, A.C., Jacobsen, E., and Feenstra, W.J. 1985. Effect of cold hardening, wilting and exogenously applied proline on leaf proline content and frost tolerance of several genotypes of solanum. Physiologia Plantarum 64: 230-237.