Arabidopsis'te Dawdle geninin FHA alanını ve C-terminal bölgesini kapsayan nokta mutasyonlarının fenotipik karakterizasyonu

Öz

Arabidopsis thaliana'daki gen fonksiyon kaybı alellerinin analizi, DAWDLE (DDL) adı verilen At3G20550 geninde bir mutasyonu ortaya çıkarmış ve DDL genindeki bu mutasyonun pleiotropik fenotiplere neden olduğu ve birçok mikro RNA seviyesini de düşürdüğü rapor edilmiştir. DAWDLE geni, prokaryot ve ökaryotlarda önemli hücresel fonksiyonlara sahip birçok proteinde de bulunan Çatal Başlı-İlişkili Bölge (FHA) alanına sahip bir proteini kodlar. Ancak, DDL proteinin FHA alanının ve C-terminal bölgesinin bu genin işlevi için gerekli olup olmadığı tam olarak bilinmemektedir. Bu çalışmada, DDL geninin FHA alanı ve C-terminal bölgesini kapsayan nokta mutasyonlarının fenotipik karakterizasyonu yapılarak her iki bölgenin DDL fonksiyonunda rolü olup olmadığı analiz edilmiştir. Arabidopsis'in Columbia erecta-105 yabanıl tipinde ‘Genomda Hedefli İndüklenmiş Lokal Lezyonlar’ (Tilling) hat taraması yapılarak DDL geninin FHA ve C-terminal bölgesini kapsayan nokta mutasyonları tespit edilmiş, ardından bu nokta mutasyonları taşıyan homozigot mutantlar belirlenerek bitki boyu, hipokotil ve kök uzunluğu ve tohum sayısı seviyesinde fenotipik olarak karakterize edilmiştir. Mutantların fenotipik analizi, farklı bitki organlarında değişen derecelerde benzer ddl fenotiplerini ortaya çıkarmıştır. Tiller mutant hatlarının tohum sayısındaki düşüş, kök, hipokotil ve gövde uzunluklarındaki kısalma, Arabidopsis'te FHA ve C-terminal bölgesinin DDL geninin fonksiyonu için önemli olabileceğini ortaya koymuştur. Anahtar Kelimeler: Dawdle, Çatal Başlı-İlişkili Bölge, Genomda Hedefli İndüklenmiş Lokal Lezyonlar, Etil Metan Sülfonat, Arabidopsis

Anahtar Kelimeler

• Dawdle
• Çatal Başlı İlişkili Bölge
• Genomda Hedeflş İndüklenmiş Lokal Lezyonlar
• Etil Metan Sülfonat
• Arabidopsis

Phenotypic characterization of point mutations spanning FHA domain and C-terminal region of Dawdle gene in Arabidopsis

Öz

The screening analysis of loss-of-function alleles in Arabidopsis thaliana revealed a mutation in the At3G20550 gene, called DAWDLE (DDL). The mutation in the DDL gene causes pleiotropic phenotypes and reduced the levels of several microRNAs. The DAWDLE gene encodes a protein with a Fork Head-Associated (FHA) domain, found in large range of proteins with significant cellular processes in prokaryotes and eukaryotes. However, it is not completely known whether the FHA domain and C-terminal region of the DDL are necessary for its function. The aim of this study was to determine the function of both regions by conducting a phenotypic analysis of point mutations spanning the FHA domain and C-terminal region in DDL Targeted Induced Local Lesions IN Genome (Tilling) screen was performed in the Columbia erecta-105 background of Arabidopsis resulting in point mutations spanning both regions of DDL. The mutants were phenotypically characterized. Height of the plant, hypocotyl and root length, and fertility were measured. Phenotypic analyses of the mutants revealed ddl phenotypes of varying degrees in different organs. Reduction in fertility and shortening in root, hypocotyl and stem lengths of the Tiller mutant lines suggest that the FHA domain and C-terminal region may require for DDL function in Arabidopsis. Key words: Dawdle, Fork Head-Associated Domain, Targeted Induced Local Lesions in Genome, Ethyl Methane Sulfonate, Arabidopsis

Anahtar Kelimeler

• Dawdle
• Fork Head-Associated Domain
• Targeted Induced Local Lesions in Genome
• Ethyl Methane Sulfonate
• Arabidopsis

Kaynakça

1. Bartel, D.P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2), 281-297. https://doi.org/10.1016/s0092-8674(04)00045-5
2. Bartlett, J. M. (2003). Technical notes for the detection of nucleic acids. Methods in Molecular Biology, 226, 65-76.https://doi.org/10.1385/1-59259-384-4:65
3. Bell, C. J., & Ecker, J. R. (1994). Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics,19(1),137-144. https://doi.org/10.1006/geno.1994.1023
4. Burkart-Waco, D., Josefsson, C., Dilkes, B. P., Kozloff, N., Torjek, O., Meyer, R. C., Altmann, T., & Comai, L. (2011). Hybrid Incompatibility in Arabidopsis Is Determined by a Multiple-Locus Genetic Network. Plant Physiology, 158, 801 - 812.
5. Chen, X. (2005). MicroRNA biogenesis and function in plants. FEBS Letters, 579(26), 5923-5931. https://doi.org/10.1016/j.febslet.2005.07.071
6. Dellaporta, S. L., Wood, J. P. A., & Hicks, J. B. (2007). A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter, 1, 19-21.
7. Durocher, D., Henckel, J., Fersht, A. R., & Jackson, S. P. (1999). The FHA domain is a modular phosphopeptide recognition motif. Molecular Cell, 4(3), 387-394.
8. Durocher, D., Smerdon, S. J., Yaffe, M. B., & Jackson, S. P. (2000a). The FHA domain in DNA repair and checkpoint signaling. Cold Spring Harbor Symposia on Quantitative Biology,65,423-431. https://doi.org/10.1101/sqb.2000.65.423

9. Durocher, D., Taylor, I. A., Sarbassova, D., Haire, L. F., Westcott, S. L., Jackson, S. P., Smerdon, S. J., & Yaffe, M. B.(2000b). The molecular basis of FHAdomain:phosphopeptide binding specificity andimplications for phospho-dependent signalingmechanisms. Molecular Cell, 6(5), 1169-1182.https://doi.org/10.1016/s1097-2765(00)00114-3
10. Greene, E. A., Codomo, C. A., Taylor, N. E., Henikoff, J. G., Till, B. J., Reynolds, S. H., Enns, L. C., Burtner, C., Johnson, J. E., Odden, A. R., Comai, L., & Henikoff, S. (2003). Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics,164(2),731-740. https://doi.org/10.1093/genetics/164.2.731
11. Hammet, A., Pike, B. L., Mitchelhill, K. I., Teh, T., Kobe, B., House, C. M., Kemp, B. E., & Heierhorst, J. (2000). FHA domain boundaries of the dun1p and rad53p cell cycle checkpoint kinases. FEBS Letters, 471(2-3), 141-146. https://doi.org/10.1016/s0014-5793(00)01392-2
12. Huck, N., Moore, J. M., Federer, M., & Grossniklaus, U. (2003). The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception. Development,130(10),2149-2159. https://doi.org/10.1242/dev.00458
13. Karunarathna, N. L., Patiranage, D. S. R., Harloff, H. J., Sashidhar, N., & Jung, C. (2021). Genomic background selection to reduce the mutation load after random mutagenesis. Scientific Reports, 11, 19404. https://doi.org/10.1038/s41598-021-98934-5
14. Kim, M., Ahn, J. W., Song, K., Paek, K. H., & Pai, H. S. (2002). Forkhead-associated domains of the tobacco NtFHA1 transcription activator and the yeast Fhl1 forkhead transcription factor are functionally conserved. Journal of Biological Chemistry, 277(41), 38781-38790. https://doi.org/10.1074/jbc.M201559200
15. Kurihara, Y., & Watanabe, Y. (2004). Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proceedings of the National Academy of Sciences, 101(34), 12753-12758.
16. Liao, H., Byeon, I. J., & Tsai, M. D. (1999). Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53. Journal of Molecular Biology,294(4),1041-1049. https://doi.org/10.1006/jmbi.1999.3313
17. Mahajan, A., Yuan, C., Lee, H., Chen, E. S., Wu, P. Y., & Tsai, M.D. (2008). Structure and function of thephosphothreonine-specific FHA domain. ScienceSignaling,1(51),re12.https://doi.org/10.1126/scisignal.151re12
18. Martin, B., Ramiro, M., Martinez-Zapater, J. M., & Alonso-Blanco, C. (2009). A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis. BMC Plant Biology, 9, 147. https://doi.org/10.1186/1471-2229-9-147
19. Matthews, C.R. (1987). Effect of point mutations on the folding of globular proteins. Methods in Enzymology, 154, 498-511.
20. McCallum, C. M., Comai, L., Greene, E. A., & Henikoff, S. (2000). Targeted screening for induced mutations. Nature Biotechnology, 18(4), 455-457. https://doi.org/10.1038/74542
21. Michaels, S. D., & Amasino, R. M. (1998). A robust method for detecting single-nucleotide changes as polymorphic markers by PCR. The Plant Journal, 14(3), 381-385. https://doi.org/10.1046/j.1365-313x.1998.00123.x
22. Morris, E. R., Chevalier, D., & Walker, J. C. (2006). DAWDLE, a forkhead-associated domain gene, regulates multiple aspects of plant development. Plant Physiology, 141(3), 932-941. https://doi.org/10.1104/pp.106.076893
23. Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., & Erlich, H.(1992). Specific enzymatic amplification of DNA invitro: the polymerase chain reaction. Biotechnology,24,17-27.http://www.ncbi.nlm.nih.gov/pubmed/1422010
24. Murashige, T., & Skoog, F. K. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15, 473-497.
25. Narayanan, L., Mukherjee, D., Zhang, S., Yu, B., & Chevalier, D.(2014). Mutational Analyses of a Fork HeadAssociated Domain Protein, DAWDLE, in Arabidopsisthaliana. American Journal of Plant Sciences, 5, 2811-2822.
26. Neff, M. M., Neff, J. D., Chory, J., & Pepper, A. E. (1998). dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. The Plant Journal,14(3),387-392. https://doi.org/10.1046/j.1365-313x.1998.00124.x
27. Neff, M. M., Turk, E., & Kalishman, M. (2002). Web-based primer design for single nucleotide polymorphism analysis. Trends in Genetics, 18(12), 613-615. https://doi.org/10.1016/s0168-9525(02)02820-2
28. Pandey, P., Panday, S. K., Rimal, P., Ancona, N., & Alexov, E.(2023). Predicting the Effect of Single Mutations on Protein Stability and Binding with Respect to Types ofMutations. International Journal of Molecular Sciences,24(15), 12073.https://doi.org/10.3390/ijms241512073
29. Schauer, S. E., Jacobsen, S. E., Meinke, D. W., & Ray, A. (2002). DICER-LIKE1: blind men and elephants in Arabidopsis development. Trends in Plant Science, 7(11),487-491. https://doi.org/10.1016/s1360-1385(02)02355-5
30. Sun, Z., Hsiao, J., Fay, D. S., & Stern, D. F. (1998). Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint. Science, 281(5374), 272-274. https://doi.org/10.1126/science.281.5374.272
31. TAIR Seq Viewer. (2021, 10 March). http://www.seqviewer.arabidopsis.org
32. Till, B. J., Colbert, T., Tompa, R., Enns, L. C., Codomo, C. A., Johnson, J. E., Reynolds, S. H., Henikoff, J. G., Greene, E. A., Steine, M. N., Comai, L., & Henikoff, S. (2003). High-throughput TILLING for functional genomics. Methods in Molecular Biology, 236, 205-220. https://doi.org/10.1385/1-59259-413-1:205
33. Till, B. J., Reynolds, S. H., Greene, E. A., Codomo, C. A., Enns, L.C., Johnson, J. E., Burtner, C., Odden, A. R., Young, K.,Taylor, N. E., Henikoff, J. G., Comai, L., & Henikoff, S.(2003). Large-scale discovery of induced pointmutations with high-throughput TILLING. GenomeResearch,13(3),524-530.https://doi.org/10.1101/gr.977903
34. Yu, B., Bi, L., Zheng, B., Ji, L., Chevalier, D., Agarwal, M., Ramachandran, V., Li, W., Lagrange, T., Walker, J. C., & Chen, X. (2008). The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proceedings of the National Academy of Sciences,105(29),10073-10078. https://doi.org/10.1073/pnas.0804218105
35. Zhang, S., Dou, Y., Li, S., Ren, G., Chevalier, D., Zhang, C., & Yu, B. (2018). DAWDLE Interacts with DICER-LIKE Proteins to Mediate Small RNA Biogenesis. Plant Physiology,177(3),1142-1151. https://doi.org/10.1104/pp.18.00354