Deep sequencing of wheat sRNA transcriptome reveals distinct temporal expression pattern of miRNAs in response to heat, light and UV

Abstract

Understanding of plant adaptation to abiotic stresses has implications in plant breeding, especially in the context of climate change. MicroRNAs (miRNAs) and short interfering RNAs play a crucial role in gene regulation. Here, wheat plants were exposed to one of the following stresses: continuous light, heat or ultraviolet radiations over five consecutive days and leaf tissues from three biological replicates were harvested at 0, 1, 2, 3, 7 and 10 days after treatment (DAT). A total of 72 small RNA libraries were sequenced on the Illumina platform generating ~524 million reads corresponding to ~129 million distinct tags from which 232 conserved miRNAs were identified. The expression levels of 1, 2 and 79 miRNAs were affected by ultraviolet radiation, continuous light and heat, respectively. Approximately 55% of the differentially expressed miRNAs were downregulated at 0 and 1 DAT including miR398, miR528 and miR156 that control mRNAs involved in activation of signal transduction pathways and flowering. Other putative targets included histone variants and methyltransferases. These results suggest a temporal miRNA-guided post-transcriptional regulation that enables wheat to respond to abiotic stresses, particularly heat. Designing novel wheat breeding strategies such as regulatory gene-based marker assisted selection depends on accurate identification of stress induced miRNAs.

Figure 1. Overview of small RNA profile.

(a) Size distribution of raw reads after size selection with Geneious. (b) Size distribution of distinct tags prior to removal of ncRNA and chloroplast sequences. (c) Distribution of 21 nt-long distinct tags by treatment. (d) Distribution of 24 nt-long distinct tags by treatment. The bars represent the mean and standard deviation from three biological replicates.

Figure 2. Conserved miRNA families.

(a) Distribution of family members and isomiRs assigned to a subset of 31 mature miRNA families with distinct unambiguous precursor evidence. (b) Relative abundance of different size classes of the same 31 mature miRNA families.

Figure 3. Differential expression of miRNAs following stress treatments.

(a) Venn diagram representing the number of the miRNAs differentially expressed in response to heat, light and UV stresses. (b) Heat map of differentially expressed miRNAs in response to heat stress. The green color represents over expressed miRNAs while red is for under expressed miRNAs. The dendrogram at the top represents the clustering of the control (C) and heat (H) treatments at the six sampling time points of 0, 1, 2, 3, 7 and 10 days after treatment (DAT) labeled at the bottom.

Figure 4. Three examples of Cytoscape networks of differentially expressed miRNAs and their targets.

MiRNAs are in purple boxes in the middle of the networks and their targets are identified by their UniProtKB ID in the surrounding blue boxes. The green boxes identify the mRNAs predicted to be targeted by more than one miRNA. Targets addressed in the discussion are marked with blue arrows.

Figure 5. STRING network view of known and predicted interaction partners.

The miRNA targets were used as query to search previously annotated proteins. (a) Maize (Zea mays), and (b) Rice (Oryza sativa). The confidence view screen shows the known and predicted protein-protein interactions where stronger associations are represented by thicker lines.