Table 1.
Stress responsive microRNAs and their response to different abiotic treatments in various plant species.
Table 2.
Summary of putative miRNA hairpins in durum wheat small RNA libraries.
Fig 1.
Heat-map showing differential expression patterns of conserved miRNAs between different genotypes revealed by high-throughput sequencing.
The colour scale is based on the log2 value of the fold-change of the water deficit stress tolerant variety (Tamaroi or Yawa) libraries compared to the water deficit stress sensitive variety (EGA Bellaroi or Tjilkuri) libraries. Log2 value = log2 (RPM of miRNA reads in Tamaroi library/RPM of miRNA reads in EGA Bellaroi library) or log2 (RPM of miRNA reads in Yawa library/RPM of miRNA reads in Tjilkuri library). The red colour indicates that the miRNA was more abundant in the water deficit stress sensitive variety; while the green colour indicates that the miRNA was more abundant in the water deficit stress tolerant variety. CG = Control group; WG = Water deficit stress group; FL = Flag leaf samples; H = Head samples; Be = EGA Bellaroi; Ta = Tamaroi; Tj = Tjilkuri; Ya = Yawa; Ath = Arabidopsis thaliana; Bdi = Brachypodium distachyon; Hvu = Hordeum vulgare; Gma = Glycine max; Osa = Oryza sativa; Sbi = Sorghum bicolor; Tae = Triticum aestivum; Zma = Zea mays.
Table 3.
Genotype-specific durum miRNAs showed four different regulation patterns to water deficit stress.
Fig 2.
Heat-map showing differential expression patterns of conserved miRNAs between different tissues revealed by high-throughput sequencing.
The colour scale is based on the log2 value of the fold-change of the developing head libraries compared to the flag leaf libraries in four durum genotypes under different water treatments. Log2 value = log2 (RPM of miRNA reads in head libraries/RPM of miRNA reads in flag leaf libraries). The red colour indicates that the miRNA was more abundant in the flag leaf libraries; while the green colour indicates that the miRNA was more abundant in the developing head libraries. CG = Control group; WG = Water deficit stress group; Be = EGA Bellaroi; Ta = Tamaroi; Tj = Tjilkuri; Ya = Yawa; Ath = Arabidopsis thaliana; Bdi = Brachypodium distachyon; Hvu = Hordeum vulgare; Gma = Glycine max; Osa = Oryza sativa; Sbi = Sorghum bicolor; Tae = Triticum aestivum; Zma = Zea mays.
Table 4.
Durum miRNAs showed tissue-specific expression profiles regardless of water deficit stress.
Fig 3.
Heat-map showing expression patterns of water deficit stress responsive conserved miRNAs revealed by high-throughput sequencing.
The colour scale is based on the log2 value of the fold-change of the water deficit stress treatment libraries compared to the control treatment libraries in four durum genotypes. Log2 value = log2 (RPM of miRNA reads in water deficit stress libraries/RPM of miRNA reads in to control libraries). The red colour indicates that the miRNA was more abundant in the control libraries; while the green colour indicates that the miRNA was more abundant in the water deficit treatment libraries. CG = Control group; WG = Water deficit stress group; FL = Flag leaf samples; H = Head samples; Be = EGA Bellaroi; Ta = Tamaroi; Tj = Tjilkuri; Ya = Yawa; Ath = Arabidopsis thaliana; Bdi = Brachypodium distachyon; Hvu = Hordeum vulgare; Gma = Glycine max; Osa = Oryza sativa; Sbi = Sorghum bicolor; Tae = Triticum aestivum; Zma = Zea mays.
Fig 4.
Venn diagram of all differentially expressed conserved microRNAs identified through Approach #1.
The number of microRNAs that were differentially abundant in each category is indicated. A total of 57 conserved miRNAs were identified as being responsive to water deficit stress, as well as being differentially abundant across different genotypes and tissue types.
Fig 5.
A schematic representation displaying the breakdown of water deficit stress responsive miRNA hairpins identified.
A total of 16 hairpins have a significant tolerance × treatment interaction term and contain at least one perfect alignment to a known mature miRNA, while five hairpins have a significant tolerance × treatment interaction term but do not contain a perfect alignment to any known mature miRNAs.
Fig 6.
Expression profiles of stress responsive miRNA hairpins showing inverted regulatory patterns between stress tolerant/sensitive genotypes.
In (A) 16 conserved miRNA hairpins representing 11 conserved miRNAs are shown, while in (B) five novel miRNA hairpins representing four conserved miRNAs are displayed. The log2 value of normalised reads for each miRNA hairpin is represented as counts per million (CPM). ■ = Control group; □ = Water deficit stress group. Tae = Triticum aestivum; Ata = Aegilops tauschii; Bdi = Brachypodium distachyon; Zma = Zea mays; Ccl = Citrus clementina; Aqc = Aquilegia coerulea; Stu = Solanum tuberosum; Vvi = Vitis vinifera; Cme = Cucumis melo; Aty = Arachis hypogaea; Tolerant = Stress tolerant genotypes (Tamaroi and Yawa); Sensitive = Stress sensitive genotypes (EGA Bellaroi and Tjilkuri); I and II denotes two different hairpins representing the same conserved miRNA.
Fig 7.
Expression analysis of stress responsive miRNA candidates by qPCR in four durum wheat genotypes.
GAPDH was used as an endogenous control. The fold change is shown as a log2 value of miRNA expression in the water deficit libraries/miRNA expression in the control libraries. FL = Flag leaf samples; H = Head samples; Be = EGA Bellaroi; Ta = Tamaroi; Tj = Tjilkuri; Ya = Yawa; Ath = Arabidopsis thaliana; Bdi = Brachypodium distachyon; Gma = Glycine max; Osa = Oryza sativa; Ttu = Triticum turgidum; Zma = Zea mays.
Fig 8.
Categorisation of predicted targets of four novel stress responsive miRNAs using Gene Ontology (GO) terms.
Pie charts representing different GO categories are based on the number of target sequences enriched in each GO term. GO terms at level 8 are used for (A) cellular component categorisation. GO terms at level 3 are used in categorisation for (B) Molecular function, and (C) Biological processes. The percentage of each GO term is based on the number of targets enriched for that term relative to the total number of targets in each category. The GO level represents the position of a GO term in the GO hierarchy. The level of a GO term is the number of GO terms between that term and the Root Term of the Ontology.