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.
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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.
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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).
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13258_2020_964_MOESM1_ESM.pptx
Figure 1. Sequence alignment of BR-responsive miRNAs and their targets. The sequences of miRNA target sites and miR156g (a), miR398a (b), and miR398a-5p (c) were aligned. Lines and circles indicate a perfect matches and G:U pairs, respectively. (PPTX 36 kb)
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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
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DOI: https://doi.org/10.1007/s13258-020-00964-2