The phytohormone cytokinin regulates diverse aspects of plant growth and development, probably through context-dependent transcriptional regulation that relies on a dynamic interplay between regulatory proteins and chromatin. We employed the assay for transposase accessible chromatin with sequencing to profile changes in the chromatin landscape of Arabidopsis roots and shoots in response to cytokinin. Our results reveal differentially accessible chromatin regions indicative of dynamic regulation in response to cytokinin. These changes in chromatin occur preferentially upstream of cytokinin-regulated genes. The changes also largely overlap with binding sites for the type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs), transcription factors that mediate the primary response to cytokinin. Furthermore, the type-B ARRs were found to be necessary for the changes in chromatin state in response to cytokinin. Last, we identified context-dependent responses by comparing root and shoot profiles. This study provides new insight into the dynamics between cytokinin and chromatin with regard to directing transcriptional programmes and how cytokinin mediates its pleiotropic effects.
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The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.The raw and processed RNA-seq and ATAC-seq data described in this study have been deposited to the NCBI Short Read Archive (SRA) database under PRJNA415015 and Gene Expression Omnibus under Series GSE116287,respectively.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Asensi-Fabado, M. A., Amtmann, A. & Perrella, G. Plant responses to abiotic stress: the chromatin context of transcriptional regulation. Biochim. Biophys. Acta 1860, 106–122 (2017).
Kieber, J. J. & Schaller, G. E. Cytokinins. Arabidopsis Book 12, e0168 (2014).
Wulfetange, K. et al. The cytokinin receptors of Arabidopsis are located mainly to the endoplasmic reticulum. Plant Physiol. 156, 1808–1818 (2011).
Caesar, K. et al. Evidence for the localization of the Arabidopsis cytokinin receptors AHK3 and AHK4 in the endoplasmic reticulum. J. Exp. Bot. 62, 5571–5580 (2011).
Punwani, J. A., Hutchison, C. E., Schaller, G. E. & Kieber, J. J. The subcellular distribution of the Arabidopsis histidine phosphotransfer proteins is independent of cytokinin signaling. Plant J. 62, 473–482 (2010).
Hutchison, C. E. et al. The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling. Plant Cell 18, 3073–3087 (2006).
Hwang, I., Sheen, J. & Muller, B. Cytokinin signaling networks. Annu. Rev. Plant. Biol. 63, 353–380 (2012).
Zubo, Y. O. et al. Cytokinin induces genome-wide binding of the type-B response regulator ARR10 to regulate growth and development in Arabidopsis. Proc. Natl Acad. Sci. USA 114, E5995–E6004 (2017).
Cuvier, O. & Fierz, B. Dynamic chromatin technologies: from individual molecules to epigenomic regulation in cells. Nat. Rev. Genet. 18, 457–472 (2017).
Spitz, F. & Furlong, E. E. Transcription factors: from enhancer binding to developmental control. Nat. Rev. Genet. 13, 613–626 (2012).
Voss, T. C. & Hager, G. L. Dynamic regulation of transcriptional states by chromatin and transcription factors. Nat. Rev. Genet. 15, 69–81 (2014).
Bhargava, A. et al. Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis. Plant Physiol. 162, 272–294 (2013).
Brenner, W. G. & Schmulling, T. Summarizing and exploring data of a decade of cytokinin-related transcriptomics. Front. Plant Sci. 6, 29 (2015).
Brenner, W. G., Ramireddy, E., Heyl, A. & Schmulling, T. Gene regulation by cytokinin in Arabidopsis. Front. Plant Sci. 3, 8 (2012).
Brenner, W. G. & Schmulling, T. Transcript profiling of cytokinin action in Arabidopsis roots and shoots discovers largely similar but also organ-specific responses. BMC Plant Biol. 12, 112 (2012).
Wang, J. et al. Cytokinin signaling activates WUSCHEL expression during axillary meristem initiation. Plant Cell 29, 1373–1387 (2017).
Furuta, K. et al. The CKH2/PKL chromatin remodeling factor negatively regulates cytokinin responses in Arabidopsis calli. Plant Cell Physiol. 52, 618–628 (2011).
Efroni, I. et al. Regulation of leaf maturation by chromatin-mediated modulation of cytokinin responses. Dev. Cell 24, 438–445 (2013).
Fry, C. J. & Farnham, P. J. Context-dependent transcriptional regulation. J. Biol. Chem. 274, 29583–29586 (1999).
McKay, D. J. & Lieb, J. D. A common set of DNA regulatory elements shapes Drosophila appendages. Dev. Cell 27, 306–318 (2013).
Daugherty, A. C. et al. Chromatin accessibility dynamics reveal novel functional enhancers in C. elegans. Genome Res. 27, 2096–2107 (2017).
Uyehara, C. M. et al. Hormone-dependent control of developmental timing through regulation of chromatin accessibility. Genes Dev. 31, 862–875 (2017).
Sullivan, A. M. et al. Mapping and dynamics of regulatory DNA and transcription factor networks in A. thaliana. Cell Rep. 8, 2015–2030 (2014).
Pajoro, A. et al. Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development. Genome Biol. 15, R41 (2014).
Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).
Xie, M. et al. A B-ARR-mediated cytokinin transcriptional network directs hormone cross-regulation and shoot development. Nat. Commun. 9, 1604 (2018).
Maher, K. A. et al. Profiling of accessible chromatin regions across multiple plant species and cell types reveals common gene regulatory principles and new control modules. Plant Cell 30, 15–36 (2018).
Sijacic, P., Bajic, M., McKinney, E. C., Meagher, R. B. & Deal, R. B. Changes in chromatin accessibility between Arabidopsis stem cells and mesophyll cells illuminate cell type-specific transcription factor networks. Plant J. 94, 215–231 (2018).
Lu, Z., Hofmeister, B. T., Vollmers, C., DuBois, R. M. & Schmitz, R. J. Combining ATAC-seq with nuclei sorting for discovery of cis-regulatory regions in plant genomes. Nucleic Acids Res. 45, e41 (2017).
Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).
Shen, L. et al. diffReps: detecting differential chromatin modification sites from ChIP-seq data with biological replicates. PLoS ONE 8, e65598 (2013).
Sullivan, A. M., Bubb, K. L., Sandstrom, R., Stamatoyannopoulos, J. A. & Queitsch, C. DNase I hypersensitivity mapping, genomic footprinting, and transcription factor networks in plants. Curr. Plant Biol. 3-4, 40–47 (2015).
Yu, G., Wang, L. G. & He, Q. Y. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 31, 2382–2383 (2015).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
D’Agostino, I. B., Deruere, J. & Kieber, J. J. Characterization of the response of the Arabidopsis response regulator gene family to cytokinin. Plant Physiol. 124, 1706–1717 (2000).
Dello Ioio, R. et al. A genetic framework for the control of cell division and differentiation in the root meristem. Science 322, 1380–1384 (2008).
Schaller, G. E., Bishopp, A. & Kieber, J. J. The yin-yang of hormones: cytokinin and auxin interactions in plant development. Plant Cell 27, 44–63 (2015).
Street, I. H. et al. Cytokinin acts through the auxin influx carrier AUX1 to regulate cell elongation in the root. Development 143, 3982–3993 (2016).
Steiner, E. et al. The Arabidopsis O-linked N-acetylglucosamine transferase SPINDLY interacts with class I TCPs to facilitate cytokinin responses in leaves and flowers. Plant Cell 24, 96–108 (2012).
Simonini, S. & Kater, M. M. Class I BASIC PENTACYSTEINE factors regulate HOMEOBOX genes involved in meristem size maintenance. J. Exp. Bot. 65, 1455–1465 (2014).
Shanks, C. M. et al. Role of BASIC PENTACYSTEINE transcription factors in a subset of cytokinin signaling responses. Plant J. 95, 458–473 (2018).
Reyes-Olalde, J. I. et al. The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium. PLoS Genet. 13, e1006726 (2017).
Ohashi-Ito, K. et al. A bHLH complex activates vascular cell division via cytokinin action in root apical meristem. Curr. Biol. 24, 2053–2058 (2014).
Cluis, C. P., Mouchel, C. F. & Hardtke, C. S. The Arabidopsis transcription factor HY5 integrates light and hormone signaling pathways. Plant J. 38, 332–347 (2004).
Dobisova, T. et al. Light controls cytokinin signaling via transcriptional regulation of constitutively active sensor histidine kinase CKI1. Plant Physiol. 174, 387–404 (2017).
Vandenbussche, F. et al. HY5 is a point of convergence between cryptochrome and cytokinin signalling pathways in Arabidopsis thaliana. Plant J. 49, 428–441 (2007).
Zdarska, M. et al. Illuminating light, cytokinin, and ethylene signalling crosstalk in plant development. J. Exp. Bot. 66, 4913–4931 (2015).
Argyros, R. D. et al. Type B response regulators of Arabidopsis play key roles in cytokinin signaling and plant development. Plant Cell 20, 2102–2116 (2008).
Ishida, K., Yamashino, T., Yokoyama, A. & Mizuno, T. Three type-B response regulators, ARR1, ARR10 and ARR12, play essential but redundant roles in cytokinin signal transduction throughout the life cycle of Arabidopsis thaliana. Plant Cell Physiol. 49, 47–57 (2008).
Weber, B., Zicola, J., Oka, R. & Stam, M. Plant enhancers: a call for discovery. Trends Plant Sci. 21, 974–987 (2016).
Liu, Y. et al. Genome-wide mapping of DNase I hypersensitive sites reveals chromatin accessibility changes in Arabidopsis euchromatin and heterochromatin regions under extended darkness. Sci. Rep. 7, 4093 (2017).
Shu, H., Wildhaber, T., Siretskiy, A., Gruissem, W. & Hennig, L. Distinct modes of DNA accessibility in plant chromatin. Nat. Commun. 3, 1281 (2012).
Wisecaver, J. H. et al. A global coexpression network approach for connecting genes to specialized metabolic pathways in plants. Plant Cell 29, 944–959 (2017).
Field, B. et al. Formation of plant metabolic gene clusters within dynamic chromosomal regions. Proc. Natl Acad. Sci. USA 108, 16116–16121 (2011).
Reimegard, J. et al. Genome-wide identification of physically clustered genes suggests chromatin-level co-regulation in male reproductive development in Arabidopsis thaliana. Nucleic Acids Res. 45, 3253–3265 (2017).
Yu, N. et al. Delineation of metabolic gene clusters in plant genomes by chromatin signatures. Nucleic Acids Res. 44, 2255–2265 (2016).
Nutzmann, H. W. & Osbourn, A. Regulation of metabolic gene clusters in Arabidopsis thaliana. New Phytol. 205, 503–510 (2015).
Meng, H. & Bartholomew, B. Emerging roles of transcriptional enhancers in chromatin looping and promoter-proximal pausing of RNA polymerase II. J. Biol. Chem. 293, 13786–13794 (2018).
Zaret, K. S. & Carroll, J. S. Pioneer transcription factors: establishing competence for gene expression. Genes Dev. 25, 2227–2241 (2011).
Iwafuchi-Doi, M. et al. The pioneer transcription factor FoxA maintains an accessible nucleosome configuration at enhancers for tissue-specific gene activation. Mol. Cell 62, 79–91 (2016).
Zhang, T. Q. et al. A two-step model for de novo activation of WUSCHEL during plant shoot regeneration. Plant Cell 29, 1073–1087 (2017).
Murashige, T. & Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497 (1962).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Ramirez, F., Dundar, F., Diehl, S., Gruning, B. A. & Manke, T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 42, W187–W191 (2014).
John, S. et al. Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nat. Genet. 43, 264–268 (2011).
Nicol, J. W., Helt, G. A., Blanchard, S. G. Jr, Raja, A. & Loraine, A. E. The Integrated Genome Browser: free software for distribution and exploration of genome-scale datasets. Bioinformatics 25, 2730–2731 (2009).
Quinlan, A. R. BEDTools: the Swiss-Army tool for genome feature analysis. Curr. Protoc. Bioinform. 47, 11–34 (2014).
Warnes, G. R. et al. gplots: Various R Programming Tools for Plotting Data v3.0.1 (CRAN, 2016); https://CRAN.R-project.org/package=gplots
Neph, S. et al. BEDOPS: high-performance genomic feature operations. Bioinformatics 28, 1919–1920 (2012).
Katari, M. S. et al. VirtualPlant: a software platform to support systems biology research. Plant Physiol. 152, 500–515 (2010).
Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Risso, D., Ngai, J., Speed, T. P. & Dudoit, S. Normalization of RNA-seq data using factor analysis of control genes or samples. Nat. Biotechnol. 32, 896–902 (2014).
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. 57, 289–300 (1995).
This work was supported by a grant from the National Science Foundation Plant Genome Research Program (IOS-1238051) to J.J.K. and G.E.S., and a National Science Foundation Plant Genome Research Program Initiative Postdoctoral Fellowship in Biology (1611875) to K.C.P. The authors thank the UNC High-Throughput Sequencing Facility and the Flow Cytometry Core Facility. The MoFlo XDP used in this study is funded by the North Carolina Biotech Center Institutional Support Grant 2005-IDG-1016. Thanks are also given to D. McKay, R. Deal, B. Schmitz and members of their labs for their assistance with the ATAC assay and the computational analyses.
Supplementary Figures 1–8 and Supplementary Tables 7 and 8.
Statistics of ATAC-seq libraries.
Root differential accessible regions in response to cytokinin treatment.
Statistics of RNA-seq libraries.
Root differentially expressed genes in response to cytokinin treatment.
Shoot differentially expressed genes in response to cytokinin treatment.
Shoot differential accessible regions in response to cytokinin treatment.
Primers used for ATAC-qPCR analysis.
Intersection of ARR10 binding sites.