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

So Young Park, Hallym University
Jae Han Choi, Chungnam National University
Dong Ha Oh, Louisiana State University
John C. Johnson, Louisiana State University
Maheshi Dassanayake, Louisiana State University
Dong Hoon Jeong, Hallym University
Man Ho Oh, Chungnam National University

Abstract

© 2020, The Genetics Society of Korea. 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.