Arabidopsis thaliana (L.) Heynh. YAPRAKLARINDA GUS VE GFP RAPORTÖR GENLERİ KULLANILARAK SENESENS İLE İLİŞKİLİ GENLERİN ANALİZİ

Year 2017, Volume: 18 Issue: 2, 175 - 183, 01.12.2017

Nihal Gören Sağlam

https://doi.org/10.23902/trkjnat.319185

Abstract

Yaprak senesensinin düzenlenme ve işleyiş yapısı birçok biyokimyasal ve
düzenleyici yolağın aktivasyonunu içeren kompleks bir olaydır. Daha önce
yapılan çalışmalar sonucunda, yaprak senesensinin moleküler mekanizmasını
anlamak için senesens ile ilişkili bazı genler (SAGs=Senescence Associated
Genes) belirlenmiştir. Bu çalışma ANAC018, ANAC019, MYB75 (PAP1) ve MYB2
genlerinin senesens ile ilişkili olabilme potansiyelini araştırmak için
planlanmıştır. Bu amaçla GUS (β-glukuronidaz) ve GFP (Yeşil Fluoresans Protein)
işaretleyicileri ile ANAC018, ANAC019, MYB75 (PAP1) ve MYB2 promotör genlerini
içeren Arabidopsis thaliana (L.) Heynh.
(ekotip Columbia) bitkisi kullanılmıştır. GUS ve GFP analizinde promotörün ne
zaman ve hangi hücre ya da hücre tiplerinde aktif olduğuna bakılmıştır.
Karanlığın teşvik ettiği senesens modeli oluşturulmuş ve GUS boyama ve konfokal
mikroskopi ile genlerin aktif olduğu zaman ve hücre tipleri belirlenmiştir. Elde edilen veriler ışığında ANAC018, ANAC019, MYB75 (PAP1)ve MYB2
genlerinin senesens sırasında, epidermis, epidermal hücrelerin nükleusları,
mezofil ve stoma hücrelerinde aktif oldukları belirlenerek bu genlerin yaprak
senesensinde rol oynadıkları ortaya konulmuştur. 

Keywords

Arabidopsis thaliana, GUS, GFP, senesens

Analysis of Senescence-Related Genes Expressions Using Gus and Gfp Reporter Genes in Arabidopsis thaliana (L.) Heynh. Leaves

Abstract

The
regulation and functional steps of leaf senescence is a complex phenomenon
involving the activation of many biochemical and regulatory pathways. As a
result of previous studies, some genes related to senescence (SAGs = Senescence
Associated Genes) have been identified to understand the molecular mechanism of
leaf senescence. This study was designed to investigate the
association potential of ANAC018, ANAC019, MYB75 (PAP1) and MYB2 genes with
senescence. For this purpose, GUS (β-glucuronidase) and GFP (Green
Fluorescent Protein) markers
and Arabidopsis thaliana (L.) Heynh.
(ecotype Columbia) plants containing ANAC018, ANAC019, MYB75 (PAP1), MYB2 promoter genes were used. GUS and GFP
analyses were used to determine when the promoter is active in which cells or
cell types. Dark-induced senescence model was designed and the time and the
cell types where the genes were active were determined by using GUS staining
and confocal microscopy. The results showed that ANAC018, ANAC019, MYB75
(PAP1) and MYB2 genes were active in epidermis, nucleus of epidermal cells,
mesophyll and stomata cells during senescence and these genes were found to
play a role in leaf senescence.

Keywords

Arabidopsis thaliana, GUS, GFP, senescence

References

• 1. Abe, H., Urao, T., Ito, T., Seki, M., Shinozaki, K. & Yamaguchi-Shinozaki, K. 2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. The Plant Cell, 15(1): 63-78.
• 2. Aida, M., Ishida, T., Fukaki, H., Fujisawa, H. & Tasaka, M. 1997. Genes involved in organ separation in Arabidopsis: an analysis of the cupshaped cotyledon mutant. The Plant Cell, 9: 841-857.
• 3. Baek, D., Park, H.C., Kim, M.C. & Yun, D.J. 2013. The role of Arabidopsis MYB2 in miR399f-mediated phosphate-starvation response. Plant Signaling & Behavior, 8(3): 362-73.
• 4. Balazadeh, S., Riaño-Pachón, D.M. & Mueller-Roeber, B. 2008. Transcription factors regulating leaf senescence in Arabidopsis thaliana . Plant Biology (Stuttgart), 10 (S1): 63-75.
• 5. Bhargava, A., Mansfield, S.D., Hall, H.C., Douglas, C.J. & Ellis, B.E. 2010. MYB75 functions in regulation of secondary cell wall formation in the Arabidopsis inflorescence stem. Plant Physiology, 154(3): 1428-1438.
• 6. Breeze, E., Harrison, E., McHattie, S., Hughes, L., Hickman, R., Hill, C., Kiddle, S., Kim, Y., Penfold, C., Jenkins, D., Zhang, C., Morris, K., Jenner, C., Jackson, S., Thomas, B., Tabrett, A., Legaie, R., Moore, J.D., Wild, D.L., Ott, S., Rand, D., Beynon, J., Denby, K., Mead, A. & Buchanan-Wollaston, V. 2011. High resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation. The Plant Cell, 23: 873-894.
• 7. Bu, Q., Jiang, H., Li, C.B., Zhai, Q., Zhang, J., Wu, X., Sun, J., Xie, Q. & Li, C. 2008. Role of the Arabidopsis thaliana NAC transcription factors ANAC019 and ANAC055 in regulating jasmonic acid-signaled defense responses. Cell Research, 18: 756-767.
• 8. Buchanan-Wollaston, V., Page, T., Harrison, E., Breeze, E., Lim, P.O., Nam, H.G., Lin, J.F., Wu, S.H., Swidzinski, J. & Ishizaki, K. 2005. Comparative transcriptome analysis reveals significant differences in gene expression and signalling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. The Plant Journal, 42: 567-585.
• 9. Cote, C. & Rutledge, R.G. 2003. An improved MUG fluorescent assay for the determination of GUS activity within transgenic tissue of woody plants. Plant Cell Reports, 21: 619-624.
• 10. Ding, Z., Li, S. & An, X. 2009. Transgenic expression of Myb15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana. Journal of Genetics and Genomics, 36: 17-29.
• 11. Gallagher, S.R. 1992. GUS Protocols: Using The GUS Gene As A Reporter Of Gene Expression. Academic Press, San Diego, 215 s.
• 12. Guo,Y. & Gan, S.S. 2012. Convergence and divergence in gene expression profiles induced by leaf senescence and 27 senescence-promoting hormonal, pathological and environmental stress treatments. Plant, Cell and Environment, 35: 644-655.
• 13. Guo, Y., Cai, Z. & Gan, S. 2004.Transcriptome of Arabidopsis leaf senescence. Plant, Cell and Environment, 27: 521-549.
• 14. Guo, Y. & Gan, S. 2011. AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis. Plant Physiology, 156(3): 1612-1619.
• 15. Hickman, R., Hill, C., Penfold, C.A., Breeze, E., Bowden, L., Moore, J.D. & Mead, A. 2013. A local regulatory network around three NAC transcription factors in stress responses and senescence in Arabidopsis leaves. The Plant Journal, 75(1): 26-39.
• 16. http://www.arabidopsis.org (Erişim tarihi: Nisan 2017).
• 17. Huang, C.K., Lo, P.C., Huang, L.F., Wu, S.J., Yeh, C.H. & Lu, C.A. 2015. A single-repeat MYB transcription repressor, MYBH, participates in regulation of leaf senescence in Arabidopsis. Plant Molecular Biology, 88(3): 269-286.
• 18. Jaradat, M.R., Feurtado, J.A., Huang, D., Lu, Y. & Cutler, A.J. 2013. Multiple roles of the transcription factor AtMYBR1/AtMYB44 in ABA signaling, stress responses, and leaf senescence. BMC Plant Biology, 13: 192.
• 19. Jefferson, R.A. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Molecular Biology Reports, 5: 387-405.
• 20. Jefferson, R.A. 1989.The GUS reporter gene system. Nature, 342: 837-838.
• 21. Jefferson, R.A., Kavanagh, T.A. & Bevan, M.W. 1987. GUS fusions: β-glucuronidase as a sensitive and versalite gene fusion marker in higher plants. EMBO Journal, 6: 3901-3907.
• 22. Jung, C., Shim, J.S. & Seo, J.S. 2010. Non-specific phytohormonal induction of atMYB44 and supression of jasmonate-responsive gene activation in Arabidopsis thaliana. Molecules and Cells, 29:71-76.
• 23. Karcher, S.J. 2002. Blue plants: Transgenic plants with the GUS reporter gene. Pages 29-42.In: Tested studies for laboratory teaching, Volume 23 (M. A. O’Donnell, Editor). Proceedings of the 23rd Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 392 s.
• 24. Kim, H.J., Nam, H.G. & Lim, P.O. 2016. Regulatory network of NAC transcription factors in leaf senescence. Current Opinion in Plant Biology, 33: 48-56.
• 25. Kucharewicz, W., Distelfeld, A., Bilger, W., Müller, M., Munné-Bosch, S., Hensel, G. & Krupinska, K. 2017. Acceleration of leaf senescence is slowed down in transgenic barley plants deficient in the DNA/RNA-binding protein WHIRLY1. Journal of Experimental Botany, 68(5): 983-996.
• 26. Li, Z., Peng, J., Wen, X. & Guo, H. 2013. Ethylene-insensitive3 is a senescence-associated gene that accelerates age-dependent leaf senescence by directly repressing miR164 transcription in Arabidopsis. Plant Cell, 25: 3311-3328.
• 27. Lim, P.O., Kim, H.J. & Nam, H.G. 2007. Leaf senescence. Annual Review of Plant Biology, 58: 115-136.
• 28. Lin, J.F. & Wu, S.H. 2004. Molecular events in senescing Arabidopsis leaves. The Plant Journal, 39: 612-628.
• 29. Lindemose, S., Jensen, M.K., de Velde, J.V., O’Shea, C., Heyndrickx, K.S., Workman, C.T., Vandepoele, K., Skriver, K. & Masi, F.D. 2014. A DNA-binding-site landscape and regulatory network analysis for NAC transcription factors in Arabidopsis thaliana. Nucleic Acids Research, 42: 7681-7693.
• 30. Martins, M.T.B., de Souza, W.R., Cunha, B.A.D.B., Basso, M.F., Oliveira, N.G., Vinecky, F. & Buckeridge, M.S. 2016. Characterization of sugarcane (Saccharum spp.) leaf senescence: implications for biofuel production. Biotechnology for Biofuels, 9(1): 153-157.
• 31. Neguyen H.T.K., Kim S.Y., Cho K-M., Hong J.V., Shin J.S. & Kim H.J. 2016. A Transcription Factor γMYB1 Binds to the P1BS cis -Element and Activates PLA 2 -γ Expression with its Co-Activator γMYB2. Plant Cell Physiology, 57(4): 784-797. 32. Nuruzzaman, M., Manimekalai, R., Sharoni, A.M., Satoh, K., Kondoh, H., Ooka, H. & Kikuchi, S. 2010. Genome-wide analysis of NAC transcription factor family in rice. Gene, 465(1): 30-44.
• 33. Pourtau, N., Jennings, R., Pelzer, E., Pallas, J. & Wingler, A. 2006. Effect of sugar-induced senescence on gene expression and implications for the regulation of senescence in Arabidopsis. Planta, 224(3): 556-568.
• 34. Puranik, S., Sahu, P.P., Srivastava, P.S. & Prasad, M. 2012. NAC proteins: regulation and role in stress tolerance. Trends Plant Science, 17: 369-381.
• 35. Qiu, J., Sun, S., Luo, S., Zhang, J., Xiao, X., Zhang, L., Wang, F. & Liu, S. 2014. Arabidopsis AtPAP1 transcription factor induces anthocyanin production in transgenic Taraxacum brevicorniculatum. Plant Cell Reports, 33(4): 669-680.
• 36. Rauf M., Arif M., Dortay H., Matallana-Ramirez L.P., Waters M.T., Gil Nam H., Lim P.O., Mueller-Roeber, M. & Balazadeh, S. 2013. ORE1 balances leaf senescence againts maintenance by antogonizing G2-like-mediated transcription. EMBO Reports, 14: 382-388.
• 37. Riechmann, J.L. & Ratcliffe, O.J. 2000. A genomic perspective on plant transcription factors. Current Opinion in Plant Biology, 3(5): 423-434.
• 38. Sakuraba, Y., Jeong, J., Kang, M.Y., Kim, J., Paek, N.C. & Choi, G. 2014. Phytocrome-interacting transcription factors PIF4 and PIF5 induce leaf senescence in Arabidopsis. Nature Communications, 5: 4636.
• 39. Sobieszczuk-Nowicka, E., Zmienko, A., Samelak-Czajka, A., Luczak, M., Pietrowska-Borek, M., Iorio, R., Del Duca, S., Figlerowicz, M. & Legocka, J. 2015. Dark-induced senescence of barley leaves involves activation of plastid transglutaminases. Aminoacids, 47: 825-838.
• 40. Tran, L.S.P., Nakashima, K. & Sakuma,Y. 2004. Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell, 16: 2481-2498.
• 41. Van der Graff, E., Schwake, R., Schneider, A., Desimone, M., Flugge, U.I. & Kunze, R. 2006. Transcription analysis of Arabidopsis membrane transporters and hormone pathways during developmental and induced leaf senescence. Plant Physiology, 141: 776-792.
• 42. Woo, H.R., Kim, J.H. & Kim, J. 2010. The RAV1 transcription factor positively regulates leaf senescence in Arabidopsis. Journal of Experimental Botany, 61: 3947-3957.
• 43. Yanhui, C., Xiaoyuan, Y., Kun, H., Meihua, L., Jigang, L., Zhaofeng, G. & Yunping, S. 2006. The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Molecular Biology, 60(1): 107-124.
• 44. Yu, L., Chen, H., Guan, Q., Ma, X., Zheng, X., Zou, C. & Li, Q. 2012. AtMYB2 transcription factor can interact with the CMO promoter and regulate its downstream gene expression. Biotechnolical Letter, 34 (9): 1749-1755.
• 45. Zhang, H. & Zhou, C. 2013. Signal transduction in leaf senescence. Plant Molecular Biology, 82: 539-545.
• 46. Zhang, X., Ju, H.W., Chung, M.S., Huang, P., Ahn, S.J. & Kim, C.S. 2011. The R-R-type MYB-like transcription factor, AtMYBL, is involved in promoting leaf senescence and modulates an abiotic stress response in Arabidopsis. Plant Cell Physiology, 52:138-148.
• 47. Zhang Y., Liu Z., Chen Y., He J-X. & Bi Y. 2015. Phytochrome-Interacting Factor 5 (PIF5) positively regulates dark-induced senescence and chlorophyll degradation in Arabidopsis. Plant Science, 237: 57-68.
• 48. Zhao, Y., Chan, Z., Gao, J., Xing, L., Cao, M., Yu, C. & Gong, Y. 2016. ABA receptor PYL9 promotes drought resistance and leaf senescence. Proceedings of the National Academy of Sciences, 113(7): 1949-1954.
• 49. Zheng, X.-Y., Spivey, N.W., Zeng, W., Liu, P.-P., Fu, Z.Q., Klessig, D.F., He, S.Y. & Dong, X. 2012. Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation. Cell Host Microbe, 11: 587–596.
• 50. Zheng, Z.L., Nafisi, M., Tam, A., Li, H., Crowell, D.N., Chary, S.N. & Yang, Z. 2002. Plasma membrane–associated ROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis. The Plant Cell, 14(11): 2787-2797.
• 51. Zhu, X., Chen, J., Xie, Z., Gao, J., Ren, G., Gao, S. & Kuai, B. 2015. Jasmonic acid promotes degreening via MYC2/3/4‐and ANAC019/055/072‐mediated regulation of major chlorophyll catabolic genes. The Plant Journal, 84(3): 597-610.