Transcriptionally and post-transcriptionally regulated microRNAs in heat stress response in barley

Abstract

Heat stress is one of the major abiotic factors that can induce severe plant damage, leading to a decrease in crop plant productivity. Despite barley being a cereal of great economic importance, few data are available concerning its thermotolerance mechanisms. In this work microRNAs (miRNAs) involved in heat stress response in barley were investigated. The level of selected barley mature miRNAs was examined by hybridization. Quantitative real-time PCR (RT-qPCR) was used to monitor the changes in the expression profiles of primary miRNA (pri-miRNA) precursors, as well as novel and conserved target genes during heat stress. The miRNA-mediated cleavage sites in the target transcripts were confirmed by degradome analysis and the 5' RACE (rapid amplification of cDNA ends) approach. Four barley miRNAs (miR160a, 166a, 167h, and 5175a) were found which are heat stress up-regulated at the level of both mature miRNAs and precursor pri-miRNAs. Moreover, the splicing of introns hosting miR160a and miR5175a is also heat induced. The results demonstrate transcriptional and post-transcriptional regulation of heat-responsive miRNAs in barley. The observed induction of miRNA expression is correlated with the down-regulation of the expression level of their experimentally identified new and conservative target genes.

Keywords: Barley; heat stress; microRNA; pri-miRNA; splicing; target gene..

Fig. 1.

The level of mature barley miRNA166, 160, 167, and 5175 is affected by heat. The mature miRNAs miR166, 167, 160, and 5175 (A–D, respectively) were detected by northern hybridization in control and heat conditions. The level of mature miRNAs was analysed at different time points: 3, 6, and 24h. U6 was used as a loading control. The level of miRNA in heat stress was quantified relative to that in control conditions at the respective time points tested.

Fig. 2.

Barley pri-miRNA precursors are induced by heat stress. The panels on the left represent RT–PCR analysis of the expression of pri-miR166a, 167h, 160a, and 5175a (A–D, respectively) in control and heat stress conditions; primer positions are marked by black triangles on the pri-miRNA schematic structures; miRNA and miRNA* are marked in red and blue, respectively. Ubiquitin (UBQ) amplification was used as a loading control. The charts on the right show the real-time PCR measurements of the expression level of pri-miRNA precursors during heat stress. The expression studies were performed at different time points: 3, 6, and 24h. The level of pri-miRNA in control conditions was assumed to be ‘1’, and the levels of pri-miRNA during heat stress were quantified relative to this standard; bars represent the means of three independent biological samples ±SD. M, GeneRuler 100bp Plus or 1kb Plus DNA ladders.

Fig. 3.

Heat stress affects the expression level of barley miRNA-regulated target genes. RT-qPCR analysis was performed to examine the expression level of target transcripts sliced by: miR166a, PHV (A), REV (B), and HOX9 (C); miR160a, ARF17 (D) and ARF13 (E); miR167h, ARF8 (F) and Nek5 (G); and miR5175a, ACC-like oxidase (H). mRNA:miRNA base-pairing diagrams below the graphs for PHV, AFR17, and Nek5 transcripts (A, D, and G, respectively) show the slicing sites. Arrows, along with the number of clones analysed, indicate the 5’ end of the cleaved product validated by 5’RACE.

Fig. 4.

Target plots (t-plots) for barley miRNA target detection using data from degradome libraries. t-plots show the distribution of the degradome tag reads along the full length of the target mRNA sequence. Grey arrows, along with the number of tag reads identified in the degradome libraries from 6-week- and 68-day-old barley plants, respectively, indicate the cleavage sites guided by miR166a (A, B, C), 160a (D, E), and 167h (F). The dotted line represents the average read value for the entire mRNA sequence.

Fig. 5.

An miRNA–target genes network module involved in heat stress response in barley.