Skip to main content

Genome-wide analysis of brassinosteroid responsive small RNAs in Arabidopsis thaliana

Abstract

Background

Brassinosteroids (BRs) are a class of phytohormones with important roles in regulating physiological and developmental processes. Small RNAs, including small interfering RNAs and microRNAs (miRNAs), are non-protein coding RNAs that regulate gene expression at the transcriptional and post-transcriptional levels. However, the roles of small RNAs in BR response have not been studied well.

Objective

In this study, we aimed to identify BR-responsive small RNA clusters and miRNAs in Arabidopsis. In addition, the effect of BR-responsive small RNAs on their transcripts and target genes were examined.

Methods

Small RNA libraries were constructed from control and epibrassinolide-treated seedlings expressing wild-type BRI1-Flag protein under its native promoter in the bri1-5 mutant. After sequencing the small RNA libraries, differentially expressed small RNA clusters were identified by examining the expression levels of small RNAs in 100-nt bins of the Arabidopsis genome. To identify the BR-responsive miRNAs, the expression levels of all the annotated mature miRNAs, registered in miRBase, were analyzed. Previously published RNA-seq data were utilized to monitor the BR-responsive expression patterns of differentially expressed small RNA clusters and miRNA target genes.

Results

In results, 38 BR-responsive small RNA clusters, including 30 down-regulated and eight up-regulated clusters, were identified. These differentially expressed small RNA clusters were from miRNA loci, transposons, protein-coding genes, pseudogenes and others. Of these, a transgene, BRI1, accumulates small RNAs, which are not found in the wild type. Small RNAs in this transgene are up-regulated by BRs while BRI1 mRNA is down-regulated by BRs. By analyzing the expression patterns of mature miRNAs, we have identified BR-repressed miR398a-5p and BR-induced miR156g. Although miR398a-5p is down-regulated by BRs, its predicted targets were not responsive to BRs. However, SPL3, a target of BR-inducible miR156g, is down-regulated by BRs.

Conclusion

BR-responsive small RNAs and miRNAs identified in this study will provide an insight into the role of small RNAs in BR responses in plants. Especially, we suggest that miR156g/SPL3 module might play a role in BR-mediated growth and development in Arabidopsis.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Data availability

The small RNA sequencing dataset generated in this study has been deposited at Gene Expression Omnibus (GEO) of National Center for Biotechnology Information (NCBI) under the accession number GSE149360.

References

  1. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106. https://doi.org/10.1186/gb-2010-11-10-r106

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Baulcombe DC, Dean C (2014) Epigenetic regulation in plant responses to the environment. Cold Spring Harb Perspect Biol 6:a019471. https://doi.org/10.1101/cshperspect.a019471

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Beauclair L, Yu A, Bouche N (2010) microRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis. Plant J 62:454–462. https://doi.org/10.1111/j.1365-313X.2010.04162.x

    CAS  Article  PubMed  Google Scholar 

  4. Bologna NG, Voinnet O (2014) The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev Plant Biol 65:473–503. https://doi.org/10.1146/annurev-arplant-050213-035728

    CAS  Article  PubMed  Google Scholar 

  5. Bucherl CA, Esse GW, Kruis A, Luchtenberg J, Westphal AH, Aker J, Hoek AV, Albrecht C, Borst JW, Vries SC (2013) Visualization of BRI1 and BAK1(SERK3) membrane receptor heterooligomers during brassinosteroid signaling. Plant Physiol 162:1911–1925. https://doi.org/10.1104/pp.113.220152

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Chuck G, Candela H, Hake S (2009) Big impacts by small RNAs in plant development. Curr Opin Plant Biol 12:81–86

    CAS  Article  Google Scholar 

  7. Clouse SD, Langford M, McMorris TC (1996) A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiol 111:671–678. https://doi.org/10.1104/pp.111.3.671

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Dai X, Zhao PX (2011) psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res 39(Suppl 2):W155–159. https://doi.org/10.1093/nar/gkr319

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Damodharan S, Zhao D, Arazi T (2016) A common miRNA160-based mechanism regulates ovary patterning, floral organ abscission and lamina outgrowth in tomato. Plant J 86:458–471. https://doi.org/10.1111/tpj.13127

    CAS  Article  PubMed  Google Scholar 

  10. Di Rubbo S, Irani NG, Russinova E (2011) PP2A phosphatases: the "on-off" regulatory switches of brassinosteroid signaling. Sci Signal 4:e25. https://doi.org/10.1126/scisignal.2002046

    CAS  Article  Google Scholar 

  11. Feng L, Zhang F, Zhang H, Zhao Y, Meyers BC, Zhai J (2020) An online database for exploring over 2000 arabidopsis small RNA libraries. Plant Physiol 182:685–691. https://doi.org/10.1104/pp.19.00959

    CAS  Article  PubMed  Google Scholar 

  12. Finet C, Jaillais Y (2012) Auxology: when auxin meets plant evo-devo. Dev Biol 369:19–31. https://doi.org/10.1016/j.ydbio.2012.05.039

    CAS  Article  PubMed  Google Scholar 

  13. Friedrichsen DM, Joazeiro CA, Li J, Hunter T, Chory J (2000) Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase. Plant Physiol 123:1247–1256. https://doi.org/10.1104/pp.123.4.1247

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Gandikota M, Birkenbihl RP, Hohmann S, Cardon GH, Saedler H, Huijser P (2007) The miRNA156/157 recognition element in the 3' UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings. Plant J 49:683–693

    CAS  Article  Google Scholar 

  15. Hackenberg M, Gustafson P, Langridge P, Shi BJ (2015) Differential expression of microRNAs and other small RNAs in barley between water and drought conditions. Plant Biotechnol J 13:2–13. https://doi.org/10.1111/pbi.12220

    CAS  Article  PubMed  Google Scholar 

  16. He JX, Gendron JM, Yang Y, Li J, Wang ZY (2002) The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proc Natl Acad Sci USA 99:10185–10190. https://doi.org/10.1073/pnas.152342599

    CAS  Article  PubMed  Google Scholar 

  17. Jaillais Y, Hothorn M, Belkhadir Y, Dabi T, Nimchuk ZL, Meyerowitz EM, Chory J (2011) Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor. Genes Dev 25:232–237. https://doi.org/10.1101/gad.2001911

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Jeong DH (2016) Functional diversity of microRNA variants in plants. J Plant Biol 59:303–310. https://doi.org/10.1007/s12374-016-0200-7

    CAS  Article  Google Scholar 

  19. Jeong DH, Park S, Zhai J, Gurazada SG, De Paoli E, Meyers BC, Green PJ (2011) Massive analysis of rice small RNAs: mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage. Plant Cell 23:4185–4207. https://doi.org/10.1105/tpc.111.089045

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Jeong DH, Schmidt SA, Rymarquis LA, Park S, Ganssmann M, German MA, Accerbi M, Zhai J, Fahlgren N, Fox SE, Garvin DF, Mockler TC, Carrington JC, Meyers BC, Green PJ (2013a) Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon. Genome Biol 14:R145. https://doi.org/10.1186/gb-2013-14-12-r145

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Jeong DH, Thatcher SR, Brown RS, Zhai J, Park S, Rymarquis LA, Meyers BC, Green PJ (2013b) Comprehensive investigation of microRNAs enhanced by analysis of sequence variants, expression patterns, ARGONAUTE loading, and target cleavage. Plant Physiol 162:1225–1245. https://doi.org/10.1104/pp.113.219873

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 14:787–799

    CAS  Article  Google Scholar 

  23. Kamthan A, Chaudhuri A, Kamthan M, Datta A (2015) Small RNAs in plants: recent development and application for crop improvement. Front Plant Sci 6:208. https://doi.org/10.3389/fpls.2015.00208

    Article  PubMed  PubMed Central  Google Scholar 

  24. Karimi M, Ghazanfari F, Fadaei A, Ahmadi L, Shiran B, Rabei M, Fallahi H (2016) The small-RNA profiles of almond (Prunus dulcis Mill.) reproductive tissues in response to cold stress. PLoS ONE 11:e0156519. https://doi.org/10.1371/journal.pone.0156519

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Khraiwesh B, Zhu JK, Zhu J (2012) Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants. Biochim Biophys Acta 1819:137–148. https://doi.org/10.1016/j.bbagrm.2011.05.001

    CAS  Article  PubMed  Google Scholar 

  26. Kozomara A, Griffiths-Jones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic acids Res 42:D68–73. https://doi.org/10.1093/nar/gkt1181

    CAS  Article  PubMed  Google Scholar 

  27. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858

    CAS  Article  Google Scholar 

  28. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. https://doi.org/10.1038/nmeth.1923

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862

    CAS  Article  Google Scholar 

  30. Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864. https://doi.org/10.1126/science.1065329

    CAS  Article  PubMed  Google Scholar 

  31. Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843–854

    CAS  Article  Google Scholar 

  32. Li J, Chory J (1997) A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90:929–938

    CAS  Article  Google Scholar 

  33. Li C, Zhang B (2016) MicroRNAs in control of plant development. J Cell Physiol 231:303–313. https://doi.org/10.1002/jcp.25125

    CAS  Article  PubMed  Google Scholar 

  34. Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC (2002) BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell 110:213–222. https://doi.org/10.1016/s0092-8674(02)00812-7

    CAS  Article  PubMed  Google Scholar 

  35. Li Y, Li C, Xia J, Jin Y (2011) Domestication of transposable elements into MicroRNA genes in plants. PLoS ONE 6:e19212. https://doi.org/10.1371/journal.pone.0019212

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Meng Y, Ma X, Chen D, Wu P, Chen M (2010) MicroRNA-mediated signaling involved in plant root development. Biochem Biophys Res Commun 393:345–349. https://doi.org/10.1016/j.bbrc.2010.01.129

    CAS  Article  PubMed  Google Scholar 

  37. Nam KH, Li J (2002) BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell 110:203–212. https://doi.org/10.1016/s0092-8674(02)00814-0

    CAS  Article  PubMed  Google Scholar 

  38. Naya L, Paul S, Valdes-Lopez O, Mendoza-Soto AB, Nova-Franco B, Sosa-Valencia G, Reyes JL, Hernandez G (2014) Regulation of copper homeostasis and biotic interactions by microRNA 398b in common bean. PLoS ONE 9:e84416. https://doi.org/10.1371/journal.pone.0084416

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Neilsen CT, Goodall GJ, Bracken CP (2012) IsomiRs - the overlooked repertoire in the dynamic microRNAome. Trends Genet 28:544–549. https://doi.org/10.1016/j.tig.2012.07.005

    CAS  Article  PubMed  Google Scholar 

  40. Niu J, Wang J, An J, Liu L, Lin Z, Wang R, Wang L, Ma C, Shi L, Lin S (2016) Integrated mRNA and miRNA transcriptome reveal a cross-talk between developing response and hormone signaling for the seed kernels of Siberian apricot. Sci Rep 6:35675. https://doi.org/10.1038/srep35675

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Noguchi T, Fujioka S, Choe S, Takatsuto S, Yoshida S, Yuan H, Feldmann KA, Tax FE (1999) Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids. Plant Physiol 121:743–752. https://doi.org/10.1104/pp.121.3.743

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Nova-Franco B, Iniguez LP, Valdes-Lopez O, Alvarado-Affantranger X, Leija A, Fuentes SI, Ramirez M, Paul S, Reyes JL, Girard L, Hernandez G (2015) The micro-RNA72c-APETALA2–1 node as a key regulator of the common bean-Rhizobium etli nitrogen fixation symbiosis. Plant Physiol 168:273–291. https://doi.org/10.1104/pp.114.255547

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Oh MH, Wang X, Kota U, Goshe MB, Clouse SD, Huber SC (2009) Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis. Proc Natl Acad Sci USA 106:658–663. https://doi.org/10.1073/pnas.0810249106

    Article  PubMed  Google Scholar 

  44. Oh E, Zhu JY, Bai MY, Arenhart RA, Sun Y, Wang ZY (2014) Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl. Elife. https://doi.org/10.7554/eLife.03031

    Article  PubMed  PubMed Central  Google Scholar 

  45. Oh MH, Honey SH, Tax FE (2020) The control of cell expansion, cell division, and vascular development by brassinosteroids: a historical perspective. Int J Mol Sci. https://doi.org/10.3390/ijms21051743

    Article  PubMed  PubMed Central  Google Scholar 

  46. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26. https://doi.org/10.1038/nbt.1754

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Rubio-Somoza I, Weigel D (2011) MicroRNA networks and developmental plasticity in plants. Trends Plant Sci 16:258–264. https://doi.org/10.1016/j.tplants.2011.03.001

    CAS  Article  PubMed  Google Scholar 

  48. Ryu H, Kim K, Cho H, Park J, Choe S, Hwang I (2007) Nucleocytoplasmic shuttling of BZR1 mediated by phosphorylation is essential in Arabidopsis brassinosteroid signaling. Plant Cell 19:2749–2762. https://doi.org/10.1105/tpc.107.053728

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Stief A, Altmann S, Hoffmann K, Pant BD, Scheible WR, Baurle I (2014) Arabidopsis miR156 regulates tolerance to recurring environmental stress through SPL transcription factors. Plant Cell 26:1792–1807. https://doi.org/10.1105/tpc.114.123851

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Sun Y, Han Z, Tang J, Hu Z, Chai C, Zhou B, Chai J (2013) Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide. Cell Res 23:1326–1329. https://doi.org/10.1038/cr.2013.131

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019. https://doi.org/10.1105/tpc.104.022830

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Tang W, Yuan M, Wang R, Yang Y, Wang C, Oses-Prieto JA, Kim TW, Zhou HW, Deng Z, Gampala SS, Gendron JM, Jonassen EM, Lillo C, DeLong A, Burlingame AL, Sun Y, Wang ZY (2011) PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1. Nat Cell Biol 13:124–131. https://doi.org/10.1038/ncb2151

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. Vert G, Chory J (2011) Crosstalk in cellular signaling: background noise or the real thing? Dev Cell 21:985–991. https://doi.org/10.1016/j.devcel.2011.11.006

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Wang X, Chory J (2006) Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 313:1118–1122. https://doi.org/10.1126/science.1127593

    CAS  Article  PubMed  Google Scholar 

  55. Wang ZY, Seto H, Fujioka S, Yoshida S, Chory J (2001) BRI1 is a critical component of a plasma-membrane receptor for plant steroids. Nature 410:380–383. https://doi.org/10.1038/35066597

    CAS  Article  PubMed  Google Scholar 

  56. Wang ZY, Nakano T, Gendron J, He J, Chen M, Vafeados D, Yang Y, Fujioka S, Yoshida S, Asami T, Chory J (2002) Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell 2:505–513. https://doi.org/10.1016/s1534-5807(02)00153-3

    CAS  Article  PubMed  Google Scholar 

  57. Wang X, Kota U, He K, Blackburn K, Li J, Goshe MB, Huber SC, Clouse SD (2008) Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling. Dev Cell 15:220–235. https://doi.org/10.1016/j.devcel.2008.06.011

    CAS  Article  PubMed  Google Scholar 

  58. Wang T, Chen L, Zhao M, Tian Q, Zhang WH (2011) Identification of drought-responsive microRNAs in Medicago truncatula by genome-wide high-throughput sequencing. BMC Genom 12:367. https://doi.org/10.1186/1471-2164-12-367

    CAS  Article  Google Scholar 

  59. Wu G, Poethig RS (2006) Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development 133:3539–3547

    CAS  Article  Google Scholar 

  60. Xie F, Wang Q, Sun R, Zhang B (2015) Deep sequencing reveals important roles of microRNAs in response to drought and salinity stress in cotton. J Exp Bot 66:789–804. https://doi.org/10.1093/jxb/eru437

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Professor Steven C. Huber to initiate our research at USDA-ARS, University of Illinois (Urbana-Champaign). This work was supported in part by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (No. NRF-2017R1A2B4004620), and by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Golden Seed Project, Ministry of Agriculture, Food and Rural Affairs (MAFRA) (213006-05-4-SBC30). This work was also supported by a grant from the Next-Generation BioGreen 21 Program (Project No. PJ01366801), Rural Development Administration, Republic of Korea, and by a grant from Hallym University (HRF-201409-001).

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Dong-Hoon Jeong or Man-Ho Oh.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

a

Figure 1. Sequence alignment of BR-responsive miRNAs and their targets. The sequences of miRNA target sites and miR156g (), miR398a (b), and miR398a-5p (c) were aligned. Lines and circles indicate a perfect matches and G:U pairs, respectively. (PPTX 36 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Park, S.Y., Choi, JH., Oh, DH. et al. Genome-wide analysis of brassinosteroid responsive small RNAs in Arabidopsis thaliana. Genes Genom 42, 957–969 (2020). https://doi.org/10.1007/s13258-020-00964-2

Download citation

Keywords

  • Brassinosteroid
  • Small RNA
  • MicroRNA
  • Arabidopsis thaliana