Türk Ekmeklik Buğday (T. aestivum L.) Çeşidi Gerek 79'da Kuraklığa-Duyarlı Genlerin mRNA Farklılık Gösterim Analizi İle Tanımlanması

Abstract

Buğday, dünyadaki en önemli tahıl ürünlerinden biridir. Dünyanın birçok bölgesi, gıda ve hayvan yemi kaynağı olarak buğdaya bağımlıdır. Kuraklık stresi buğdayın gelişimini olumsuz etkiler ve üretimini büyük ölçüde azaltır. Kuraklık tepkisi çok sayıda gen, transkripsiyon faktörleri, miRNA'lar, hormonlar, proteinler, kofaktörler, iyonlar ve metabolitleri içeren karmaşık bir genetik mekanizmadır. Kuraklığa dayanıklılık mekanizmalarının genetik temellerinin anlaşılması, buğdayda bu özelliğin genetik ıslahı için oldukça önemlidir. Buğday Türkiye için de önemli bir tahıl ürünüdür. Bu çalışmada, ekmeklik buğdayda (Triticum aestivum cv. Gerek 79) kuraklık stresini düzenleyen genlerin belirlenmesi amaçlanmış ve kontrol ve kuraklık stresi altındaki (%62,4 RWC) bitkilerin fide yapraklarında mRNA farklılık gösterimi (mRNA DD) kullanılarak gen ekspresyon profillemesi yapılmıştır. Karşılaştırmalı profilleme çalışması, farklı şekilde eksprese edilen toplam 20 cDNA bandı göstermiş ve bu bantlardan 10 tanesi klonlanmış ve dizilenmiştir. Düşük kaliteli okumalara sahip olanlar elenmiştir. G1 ve G2 olarak adlandırılan kalan iki cDNA'nın nükleotid dizileri benzerlik analizine tabi tutulmuştur. G1 ve G2 sırasıyla Triticum aestivum ve diğer bazı bitkilerin mor asit fosfataz mRNA dizisine ve glikosiltransferaz ailesi 92 protein benzeri dizisine yüksek derecede homoloji göstermiştir. Mor asit fosfatazların, abiyotik ve biyotik streslere karşı bitki tepkilerinde yer aldığı gösterilmiştir. Benzer şekilde, glikosiltransferazların bitkilerde hücresel homeostaz ve detoksifikasyon yollarındaki işlevlerinin yanı sıra pirinçte termotolerans üzerinde rolü olduğu bildirilmiştir. Bu raporlar ve bulgularımız, G1 ve G2 cDNA klonlarının daha fazla araştırılması için bir temel oluşturmuştur. Mor asit fosfataz ve glikosiltransferaz ailesi 92 protein benzeri diziye karşılık gelen bu gen parçalarının kuraklık koşulları altında RNA düzeyinde gen ekspresyonu değişikliklerinin araştırılması devam etmektedir. Bu genlerin ileri karakterizasyonu kuraklık tepkisindeki işlevlerinin anlaşılmasında önemli olabilir.

Keywords

• Buğday
• Kuraklık
• mRNA farklılık gösterimi
• gen ekspresyonu

Project Number

TUBITAK TOGTAG TARP/2463

Identification of Drought-Responsive Genes in Turkish Bread Wheat (T. aestivum L.) Cultivar Gerek 79 by mRNA Differential Display Analysis

Abstract

Wheat is one of the most important cereal crops in the world. Many parts of the world depend on wheat as a source of food and animal feed. Drought stress negatively affects its development and greatly reduce its production. Drought response is a complex genetic mechanism involving multiple genes, transcription factors, miRNAs, hormones, proteins, co-factors, ions, and metabolites. The understanding of genetic basis of the drought tolerance mechanisms is very important for genetic improvement of this trait in wheat. Wheat is also an important cereal crop in Turkey. In this study, it is aimed to identify drought stress-regulated genes in bread wheat (Triticum aestivum cv. Gerek 79) and gene expression profiling using mRNA differential display (mRNA DD) was performed for seedling leaves of control and drought-stressed plants (62.4% of RWC). The comparative profiling study showed a total of 20 differentially-expressed cDNA bands and 10 of them were cloned and sequenced. The inserts having poor quality reads were eliminated. The nucleotide sequences of the remaining two cDNAs named G1 and G2 were subjected to similarity analysis. G1 and G2 showed a high degree of homology to mRNA sequence of purple acid phosphatase and glycosyltransferase family 92 protein-like sequence of Triticum aestivum and some other plants, respectively. Purple acid phosphatases have been shown to be involved in plant responses to abiotic and biotic stresses. Similarly, the role of glycosyltransferases on thermotolerance has been reported in rice besides their functions in cellular homeostasis and detoxification pathways in plants. These reports and our findings have laid a foundation for further investigation of G1 and G2 cDNA clones. The investigation of differential expression of these gene fragments corresponding to purple acid phosphatase and glycosyltransferase family 92 protein-like sequence under drought conditions at the RNA level is ongoing. Further characterization of these genes could be important in understanding the functions of these gene/s in drought response.

Keywords

• Wheat
• Drought
• mRNA differential display
• gene expression

Supporting Institution

TÜBİTAK

Project Number

TUBITAK TOGTAG TARP/2463

Thanks

We sincerely thank Ömer Kaçar for his technical assistance in this study.

References

1. 1. Bray, E.A., Plant responses to water deficit. Trends Plant Sci., 1997. 2(2): p. 48–54. https://doi.org/10.1016/S1360-1385 (97)82562-9
2. 2. Gray, S.B. and S.M. Brady, Plant developmental responses to climate. Chang. Dev. Biol., 2016. 419: p. 64–77.
3. 3. McWilliam, J.R, The dimensions of drought, in Drought resistance in cereals, F.W.G. Baker, Editor. 1989, Paris: ICSU, p. 1–11.
4. 4. Budak, H., et al., From genetics to functional genomics: Improvement in drought signaling and tolerance in wheat. Front. Plant Sci,, 2015. 6: p. 1012. https://doi.org/10.3389/fpls.2015.01012
5. 5. Cushman, J.C. and H.J. Bohnert, Genomic approaches to plant stress tolerance. Current Opinion in Plant Biology, 2000. 3 (2) : p. 117–124.
6. 6. Seki, M., et al., Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses using a full-length cDNA microarray. Plant Cell, 2001. 13: p. 61–72.
7. 7. Kathiresan, A., et al., Gene expression microarrays and their application in drought stress research. Field Crops Res, 2006. 97: p. 101–110.
8. 8. Seki, M., et al., Transcriptome analysis of plant drought and salt stress response, in: Advances in molecular breeding toward drought and salt tolerant crops, M.A. Jenks, P.M. Hasegawa, S.M. Jain, Editors. 2007, Springer. Berlin. p 261–283.

9. 9. Guo, P., et al., Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage. J Exp Bot, 2009. 60: p. 3531– 3544.
10. 10. Iquebal, M.A., et al., RNAseq analysis reveals drought-responsive molecular pathways with candidate genes and putative molecular markers in root tissue of wheat. Sci Rep, 2019. 9: p. 13917. https://doi.org/10.1038/s41598-019-49915-2
11. 11. Liang, P. and A.B. Pardee, Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 1992. 257: p. 967–971.
12. 12. Tyagi, A. and A. Chandra, Isolation of stress responsive Psb A gene from rice (Oryza sativa L.) using differential display. Indian Journal of Biochemsitry and Biophysics, 2006. 43: p. 244–246.
13. 13. O’Mahony, P.J. and M.J. Oliver, Characterization of a desiccation-responsive small GTP-binding protein (Rab2) from the desiccation-tolerant grass Sporobolus stapfianus. Plant Molecular Biology, 1999. 39: p. 809–821.
14. 14. Muramoto, Y., et al., Enhanced expression of a nuclease gene in leaves of barley plants under salt stress. Gene, 1999. 234(2): p. 315–321. ISSN 0378-1119, https://doi.org/10.1016/S0378- 1119(99)00193-6
15. 15. Joshi, C.E. and H.T. Nguyen, Differential display-mediated rapid identification of different members of a multigene family, HSP16.9 in wheat. Plant Molecular Biology, 1996. 31: p. 575–584.
16. 16. Cebeci, O., et al., Differential expression of wheat transcriptomes in response to varying cadmium concentrations. Biologia Plantarum, 2008. 52 (4): p. 703–708.
17. 17. Keskin, B.C, et al., Abscisic acid regulated gene expression in bread wheat (Triticum aestivum L.) AJCS, 2020. 4(8): p. 617-625. ISSN: 1835–2707
18. 18. Turner, N.C., Techniques and experimental approaches for the measurement of plant water status. Plant and Soil, 1981. 58: 339–366. http://dx.doi.org/10.1007/BF02180062
19. 19. Verwoerd, T.C., B.M.M. Dekker, and A. Hoekema, A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acid Research, 1989. 17: p. 2362.
20. 20. Sambrook, J., E.R. Fritsch, and T. Maniatis, 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
21. 21. Hall, T.A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 1999. 41: p. 95-98. ISSN: 0261-3166
22. 22. Altschul, S.F., et al., Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research, 1997. 25: p. 3389-3402. http://dx.doi.org/10.1093/nar/25.17.3389
23. 23. Tanrivermis, H. and I. Akdogan, The use of certified seeds of improved wheat varieties in farms and the contributions of certified seed usage to enterprise economies: The case of Ankara province in Turkey. Pakistan Journal of Biological Sciences, 2007. 10 (24): p. 4339–4353. https://scialert.net /abstract/?doi=pjbs.2007.4339.4353
24. 24. Schenk, G., et al., Binuclear metal centers in plant purple acid phosphatases: Fe–Mn in sweet potato and Fe–Zn in soybean. Arch. Biochem. Biophys., 1999. 370: p. 183–189. https://doi.org/10.1006/abbi.1999.1407
25. 25. Bhadouria, J. and J. Giri, Purple acid phosphatases: roles in phosphate utilization and new emerging functions. Plant Cell Rep, 2022. 41: p. 33–51. https://doi.org/10.1007/s00299-021-02773-7
26. 26. Li, D., et al., Purple acid phosphatases of Arabidopsis thaliana. Comparative Plant Cell Reports, 2022. 41: p. 33–51.
27. 27. Zhang, Q., et al., Identification of rice purple acid phosphatases related to phosphate starvation signalling. Plant Biol., 2011. 13: p. 7–15. https://doi.org/10.1111/j.1438-8677.2010.00346.x
28. 28. Srivastava, R., et al., Identification, structure analysis, and transcript profiling of purple acid phosphatases under Pi deficiency in tomato (Solanum lycopersicum L.) and its wild relatives. Int J Biol Macromol, 2020. 165: p. 2253–2266.
29. 29. Wang, L., et al., The Arabidopsis purple acid phosphatase AtPAP10 is predominantly associated with the root surface and plays an important role in plant tolerance to phosphate limitation. Plant Physiol, 2011. 157: p. 1283–1299.
30. 30. Sun, F., et al., AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria. Plant Signal Behav., 2012. 7: p. 927–932.
31. 31. Li, C., et al., A purple acid phosphatase, GmPAP33, participates in arbuscule degeneration during arbuscular mycorrhizal symbiosis in soybean. Plant Cell Environ, 2019. 42: p. 2015–2027.
32. 32. Mehra, P., B.K. Pandey, and J. Giri, Genome-wide DNA polymorphisms in low phosphate tolerant and sensitive rice genotypes. Sci Rep, 2015. 5: p. 13090.
33. 33. Bhadouria, J., et al., Giri J., Identification of purple acid phosphatases in chickpea and potential roles of CaPAP7 in seed phytate accumulation. Sci Rep, 2017. 7: p. 1–12.
34. 34. Zhang, W., et al., An Arabidopsis purple acid phosphatase with phytase activity increases foliar ascorbate. Plant Physiol, 2008. 146: p. 431–440.
35. 35. Ravichandran, S., et al., Purple acid phosphatase 5 is required for maintaining basal resistance against Pseudomonas syringae in Arabidopsis. BMC Plant Biol, 2013. 13: p. 1–2.
36. 36. Abbasi-Vineh, M.A., M.S. Sabet, and G. Karimzadeh, Identification and functional analysis of two purple acid phosphatases AtPAP17 and AtPAP26 involved in salt tolerance in Arabidopsis thaliana, Plant. Front. Plant Sci., 2012. 11: p. 618716. https://doi.org/10.3389/fpls.2020.618716
37. 37. Reddy, C.S., Kim, K.M., James, D., Varakumar, P., Reddy, M.K., PgPAP18, a heat-inducible novel purple acid phosphatase 18-like gene (PgPAP18-like) from Pennisetum glaucum, may play a crucial role in environmental stress adaptation. Acta Physiol Plant, 2017. 39: p. 54. https://doi.org/10.1007/s11738-017-2348-2
38. 38. Liao, H., et al., GmPAP3, a novel purple acid phosphatase-like gene in soybean induced by NaCl stress but not phosphorus deficiency. Gene, 2003. 318: p. 103-111. ISSN 0378-1119. https://doi.org/10.1016/S0378-1119(03)00764-9
39. 39. Hansen, S.F., et al., Plant glycosyltransferases beyond CAZy: a perspective on DUF families. Frontiers in Plant Science, March 2012. 3 (59).
40. 40. Lim, E-K. and D.J. Bowles, A class of plant glycosyltransferases involved in cellular homeostasis. The EMBO Journal, 2004. 23: p. 2915–2922.
41. 41. Kumar, S., et al., Meta-QTLs, ortho-MQTLs, and candidate genes for thermotolerance in wheat (Triticum aestivum L.). Mol Breeding, 2021. 41(69). https://doi.org/10.1007/s11032-021-01264-7
42. 42. Dong, N.Q., et al., UDP-glucosyltransferase regulates grain size and abiotic stress tolerance associated with metabolic flux redirection in rice. Nat Commun, 2020. 11: p. 2629. https://doi.org/10.1038/s41467-020-16403-5