Indian Journal of Plant Physiology

, Volume 23, Issue 3, pp 543–556 | Cite as

Analysis of DNA methylome and transcriptome profiling following Gibberellin A3 (GA3) foliar application in Nicotiana tabacum L.

  • Raman ManoharlalEmail author
  • G. V. S. Saiprasad
  • Vinay Kaikala
  • R. Suresh Kumar
  • Ales Kovařík
Original Article


The present work investigated a comprehensive genome-wide landscape of DNA methylome and its relationship with transcriptome upon gibberellin A3 (GA3) foliar application under practical field conditions in solanaceae model, Nicotiana tabacum L. Methylated DNA Immunoprecipitation-Sequencing (MeDIP-Seq) analysis uncovered over 82% (18,456) of differential methylated regions (DMRs) in intergenic-region. Within protein-coding region, 2339 and 1685 of identified DMRs were observed in genebody- and promoter-region, respectively. Microarray study revealed 7032 differential expressed genes (DEGs) with 3507 and 3525 genes displaying up- and down-regulation, respectively. Integration analysis revealed 520 unique non-redundant annotated DMRs overlapping with DEGs. Our results indicated that GA3 induced DNA hypo- as well as hyper-methylation were associated with both gene-silencing and -activation. No complete biasness or correlation was observed in either of the promoter- or genebody-regions, which otherwise showed an overall trend towards GA3 induced global DNA hypo-methylation. Taken together, our results suggested that differential DNA methylation mediated by GA3 may only play a permissive role in regulating the gene expression.


Nicotiana tabacum GA3 Microarray DNA methylation and MeDIP-Seq 



CpG islands


Differential Expressed Genes


Differential Methylated Regions


Methylated DNA Immunoprecipitation



We would like to acknowledge Dr. C.C. Lakshmanan [Head, Corporate R&D, ITC Limited, ITC Life Science and Technology Centre (LSTC)] for his consistent support. Our appreciation is extended to Prof. Sanjeev Galande [Head, Centre of Excellence in Epigenetics, Indian Institute of Science Education and Research (IISER), Pune, India] for providing his valuable inputs on MeDIP-Seq experiment. Nucleome Informatics Private Limited, Hyderabad, India and Genotypic Technology Private Limited, Bengaluru, India are acknowledged for performing the MeDIP-Seq and microarray processing and data analysis, respectively. We would also like to acknowledge EpigenDx Hopkinton, MA, USA for carrying out tNGBS assays. Editorial and useful tips from the in-house manuscript committee of LSTC-ITC are well appreciated. Overall support from team Agriscience, ITC-LSTC is greatly acknowledged. We also acknowledge the field-workers at Northern Light Soil (NLS) region, Rajahmundry, Andhra Pradesh (A.P.), India for providing their consistent help and support during field experiments and sample collections.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflict of interest. The content of this manuscript does not necessarily reflect the views or policies of the ITC-LSTC.

Supplementary material

40502_2018_393_MOESM1_ESM.doc (121 kb)
Supplementary material 1 (DOC 121 kb)
40502_2018_393_MOESM2_ESM.ppt (2.6 mb)
Supplementary material 2 (PPT 2612 kb)
40502_2018_393_MOESM3_ESM.ppt (958 kb)
Supplementary material 3 (PPT 959 kb)
40502_2018_393_MOESM4_ESM.ppt (346 kb)
Supplementary material 4 (PPT 346 kb)
40502_2018_393_MOESM5_ESM.xls (19.9 mb)
Supplementary material 5 (XLS 20428 kb)
40502_2018_393_MOESM6_ESM.xlsx (1.3 mb)
Supplementary material 6 (XLSX 1375 kb)
40502_2018_393_MOESM7_ESM.xls (6.1 mb)
Supplementary material 7 (XLS 6274 kb)
40502_2018_393_MOESM8_ESM.xls (225 kb)
Supplementary material 8 (XLS 225 kb)
40502_2018_393_MOESM9_ESM.xls (3.2 mb)
Supplementary material 9 (XLS 3258 kb)
40502_2018_393_MOESM10_ESM.xls (13.8 mb)
Supplementary material 10 (XLS 14157 kb)
40502_2018_393_MOESM11_ESM.xlsx (608 kb)
Supplementary material 11 (XLSX 607 kb)
40502_2018_393_MOESM12_ESM.xls (14.9 mb)
Supplementary material 12 (XLS 15287 kb)
40502_2018_393_MOESM13_ESM.xlsx (673 kb)
Supplementary material 13 (XLSX 672 kb)
40502_2018_393_MOESM14_ESM.xlsx (19 kb)
Supplementary material 14 (XLSX 18 kb)


  1. Aran, D., Toperoff, G., Rosenberg, M., & Hellman, A. (2011). Replication timing-related and gene body-specific methylation of active human genes. Human Molecular Genetics, 20(4), 670–680. Scholar
  2. Ashikawa, I. (2001). Gene-associated CpG islands in plants as revealed by analyses of genomic sequences. The Plant Journal, 26(6), 617–625.CrossRefGoogle Scholar
  3. Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes & Development, 16(1), 6–21. Scholar
  4. Bujnicki, J. M., Feder, M., Radlinska, M., & Rychlewski, L. (2001). mRNA:guanine-N7 cap methyltransferases: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure-function relationships. BMC Bioinformatics, 2, 2.CrossRefGoogle Scholar
  5. Chan, S. W., Henderson, I. R., & Jacobsen, S. E. (2005). Gardening the genome: DNA methylation in Arabidopsis thaliana. Nature Reviews Genetics, 6(5), 351–360. Scholar
  6. da Huang, W., Sherman, B. T., & Lempicki, R. A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols, 4(1), 44–57. Scholar
  7. Desikan, R. S. A. H.-M., Hancock, J. T., & Neill, S. J. (2001). Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiology, 127(1), 159–172.CrossRefGoogle Scholar
  8. Edwards, R., Dixon, D. P., & Walbot, V. (2000). Plant glutathione S-transferases: enzymes with multiple functions in sickness and in health. Trends in Plant Science, 5(5), 193–198.CrossRefGoogle Scholar
  9. Exner, V., Taranto, P., Schonrock, N., Gruissem, W., & Hennig, L. (2006). Chromatin assembly factor CAF-1 is required for cellular differentiation during plant development. Development, 133(21), 4163–4172. Scholar
  10. Gao, M., Huang, Q., Chu, Y., Ding, C., Zhang, B., & Su, X. (2014). Analysis of the leaf methylomes of parents and their hybrids provides new insight into hybrid vigor in Populus deltoides. BMC Genetics, 15(Suppl 1), S8. Scholar
  11. Hardcastle, T. J. (2013). High-throughput sequencing of cytosine methylation in plant DNA. Plant Methods, 9(1), 16. Scholar
  12. Illingworth, R., Kerr, A., Desousa, D., Jorgensen, H., Ellis, P., Stalker, J., et al. (2008). A novel CpG island set identifies tissue-specific methylation at developmental gene loci. PLoS Biology, 6(1), e22. Scholar
  13. Kass, S. U., Landsberger, N., & Wolffe, A. P. (1997). DNA methylation directs a time-dependent repression of transcription initiation. Current Biology, 7(3), 157–165.CrossRefGoogle Scholar
  14. Kliebenstein, D. J., Lim, J. E., Landry, L. G., & Last, R. L. (2002). Arabidopsis UVR8 regulates ultraviolet-B signal transduction and tolerance and contains sequence similarity to human regulator of chromatin condensation 1. Plant Physiology, 130(1), 234–243. Scholar
  15. Langmead, B., & Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nature Methods, 9(4), 357–359. Scholar
  16. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., et al. (2009). The sequence alignment/map format and SAMtools. Bioinformatics, 25(16), 2078–2079. Scholar
  17. Li, X., Zhu, J., Hu, F., Ge, S., Ye, M., Xiang, H., et al. (2012). Single-base resolution maps of cultivated and wild rice methylomes and regulatory roles of DNA methylation in plant gene expression. BMC Genomics, 13, 300. Scholar
  18. Lorincz, M. C., Dickerson, D. R., Schmitt, M., & Groudine, M. (2004). Intragenic DNA methylation alters chromatin structure and elongation efficiency in mammalian cells. Nature Structural & Molecular Biology, 11(11), 1068–1075. Scholar
  19. Manoharlal, R., Saiprasad, G. V. S., Thambrahalli, A., & Madhavakrishna, K. (2018a). Dissecting the transcriptional networks underlying the gibberellin response in Nicotiana tabacum. Biologia Plantarum, 62, 647–662.CrossRefGoogle Scholar
  20. Manoharlal, R., Saiprasad, G. V. S., Ullagaddi, C., & Kovařík, A. (2018b). Gibberellin A3 (GA3) as an epigenetic determinant of global DNA hypo-methylation in tobacco. Biologia Plantarum, 62, 11–23.CrossRefGoogle Scholar
  21. Martienssen, R. A., & Colot, V. (2001). DNA methylation and epigenetic inheritance in plants and filamentous fungi. Science, 293(5532), 1070–1074. Scholar
  22. Pai, A. A., Bell, J. T., Marioni, J. C., Pritchard, J. K., & Gilad, Y. (2011). A genome-wide study of DNA methylation patterns and gene expression levels in multiple human and chimpanzee tissues. PLoS Genetics, 7(2), e1001316. Scholar
  23. Parkin, I. A., Koh, C., Tang, H., Robinson, S. J., Kagale, S., Clarke, W. E., et al. (2014). Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea. Genome Biology, 15(6), R77. Scholar
  24. Ramiro, A. R., & Barreto, V. M. (2015). Activation-induced cytidine deaminase and active cytidine demethylation. Trends in Biochemical Sciences, 40(3), 172–181. Scholar
  25. Rountree, M. R., & Selker, E. U. (1997). DNA methylation inhibits elongation but not initiation of transcription in Neurospora crassa. Genes & Development, 11(18), 2383–2395.CrossRefGoogle Scholar
  26. Schumacher, K., & Krebs, M. (2010). The V-ATPase: small cargo, large effects. Current Opinion in Plant Biology, 13(6), 724–730. Scholar
  27. Vierstra, R. D. (2012). The expanding universe of ubiquitin and ubiquitin-like modifiers. Plant Physiology, 160(1), 2–14. Scholar
  28. Yuan, Y., Qi, L., Yu, J., Wang, X., & Huang, L. (2015). Transcriptome-wide analysis of SAMe superfamily to novelty phosphoethanolamine N-methyltransferase copy in Lonicera japonica. International Journal of Molecular Sciences, 16(1), 521–534. Scholar
  29. Zemach, A., McDaniel, I. E., Silva, P., & Zilberman, D. (2010). Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science, 328(5980), 916–919. Scholar
  30. Zhang, M., Kimatu, J. N., Xu, K., & Liu, B. (2010). DNA cytosine methylation in plant development. J Genet Genomics, 37(1), 1–12. Scholar
  31. Zhang, M., Xie, S., Dong, X., Zhao, X., Zeng, B., Chen, J., et al. (2014). Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize. Genome Research, 24(1), 167–176. Scholar
  32. Zhang, X., Yazaki, J., Sundaresan, A., Cokus, S., Chan, S. W., Chen, H., et al. (2006). Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell, 126(6), 1189–1201. Scholar
  33. Zilberman, D., Gehring, M., Tran, R. K., Ballinger, T., & Henikoff, S. (2007). Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nature Genetics, 39(1), 61–69. Scholar

Copyright information

© Indian Society for Plant Physiology 2018

Authors and Affiliations

  1. 1.Corporate R&D (Agrisciences), ITC Limited, ITC Life Science and Technology Centre (LSTC)BengaluruIndia
  2. 2.Academy of Sciences of the Czech Republic, Institute of Biophysics, v.v.iBrnoCzech Republic

Personalised recommendations