Validation of miRNA-directed cleavage of predicted target mRNA using 5′ RLM- RACE.

<p>(<b>A</b>) The cleavage sites of three target genes were identified using 5′ RLM-RACE analysis. The arrows indicate 5′ termini of miRNA-guided cleavage products and the number indicates frequency of the cloned PCR products sequenced. (<b>B</b>) Tissue- and abiotic stress-specific expression profiling of validated targets of wheat miRNAs. qPCR was performed and normalization was carried out with wheat APT (Adenine Phosphoribosyl Transferase) gene. Error bars represent standard error of three biological replicates. C: control wheat seedlings; HS_35: high temperature stress at 35°C; HS_40: high temperature stress at 40°C; SS_150: salinity stress with 150 mM NaCl; SS_250: salinity stress with 250 mM NaCl; WDS_PEG: water-deficit stress imposed by 20% PEG; WDS_MAN: water-deficit stress imposed by 400 mM mannitol.</p

A Comprehensive Genome-Wide Study on Tissue-Specific and Abiotic Stress-Specific miRNAs in <i>Triticum aestivum</i>

<div><p>Productivity of wheat crop is largely dependent on its growth and development that, in turn, is mainly regulated by environmental conditions, including abiotic stress factors. miRNAs are key regulators of gene expression networks involved in diverse aspects of development and stress responses in plants. Using high-throughput sequencing of eight small RNA libraries prepared from diverse abiotic stresses and tissues, we identified 47 known miRNAs belonging to 20 families, 49 true novel and 1030 candidate novel miRNAs. Digital gene expression analysis revealed that 257 miRNAs exhibited tissue-specific expression and 74 were associated with abiotic stresses. Putative target genes were predicted for miRNAs identified in this study and their grouping into functional categories indicated that the putative targets were involved in diverse biological processes. RLM-RACE of predicted targets of three known miRNAs (miR156, miR160 and miR164) confirmed their mRNA cleavage, thus indicating their regulation at post-transcriptional level by the corresponding miRNAs. Mapping of the sequenced data onto the wheat progenitors and closely related monocots revealed a large number of evolutionary conserved miRNAs. Additional expression profiling of some of these miRNAs in other abiotic stresses underline their involvement in multiple stresses. Our findings provide valuable resource for an improved understanding of the role of miRNAs in stress tolerance as well as plant development.</p></div

Heat map representing q-PCR based expression profiling of wheat miRNAs during cold stress, nutrient stress, oxidative stress and exogenous application of various hormones.

<p>PolyA tailing of total RNA followed by cDNA synthesis and Taqman-based qPCR was employed for validation of miRNAs and normalization was carried out with wheat 5 S rRNA. GA: gibberellic acid; BS: brassinosteroids; ABA: abscisic acid; JA: methyl jasmonate; oxidative stress imposed by hydrogen peroxide (H₂O₂) and methyl viologen (MV); CS: cold stress; N: nitrogen deprivation; P: phosphorus deprivation; K: potassium deprivation; S: sulphur deprivation.</p

Analysis of digital expression of miRNAs in wheat tissue- and abiotic stress-specific libraries.

<p>(<b>A</b>) Venn diagram representing overlap of miRNA population in tissue-specific libraries viz. shoot, root, mature leaf and spikelet. (<b>B</b>) Unsupervised hierarchical cluster analysis of the normalized expression levels (TPM) of miRNAs during three abiotic stresses (high temperature, salinity and water-deficit stress). The clustering of three stress-treated samples along with control sample was performed using Pearson uncentered algorithm with an average linkage rule, to identify clusters of miRNA based on their expression levels across samples. miRNAs exhibiting ≥2 fold change in expression levels were included in this analysis. (<b>C</b>) Venn diagram representing small RNAs overlapping among tissue- and stress- specific libraries. C: control wheat seedlings; HS: high temperature stress; SS: salinity stress; WDS: water-deficit stress.</p

qPCR validation and expression analysis of wheat miRNAs mapping onto genomic sequence of ancestral species in tissues and during various stresses in wheat.

<p>PolyA tailing followed by cDNA synthesis and qPCR was employed for validation of miRNAs and the expression level of miRNAs were normalized with respect to wheat 5(A) qPCR of miRNAs in tissues; (B) Heat map representation of qPCR of miRNAs during abiotic stress and application of hormones. (HS_35: heat stress at 35°C; HS_40: heat stress at 40°C; SS_150: salinity stress with 150 mM NaCl solution; SS_250: salinity stress with 250 mM NaCl solution; WDS_PEG: water-deficit stress by 20% PEG solution; WDS_MAN: water-deficit stress by 400 mM mannitol; GA: gibberellic acid; BS: brassinosteroids; ABA: abscisic acid; JA: methyl jasmonate; oxidative stress imposed by hydrogen peroxide (H₂O₂) and methyl viologen (MV); CS: cold stress; N: nitrogen deprivation; P: phosphorus deprivation; K: potassium deprivation; S: sulphur deprivation)</p

Abiotic stress-specific expression profiling of miRNAs identified in wheat small RNA libraries by qPCR method.

<p>(<b>A</b>) known miRNAs, (<b>B</b>) true novel miRNAs. PolyA tailing of total RNA followed by cDNA synthesis and Taqman-based qPCR was employed for validation of miRNAs and normalization was carried out with wheat 5S rRNA. Error bars represent standard error of three independent biological replicates. C: control wheat seedlings; HS_35: high temperature stress at 35°C; HS_40: high temperature stress at 40°C; SS_150: salinity stress with 150 mM NaCl; SS_250: salinity stress with 250 mM NaCl; WDS_PEG: water-deficit stress imposed by 20% PEG; WDS_MAN: water-deficit stress imposed by 400 mM mannitol.</p

GO analysis of predicted targets of known and novel miRNAs.

<p>Top 5 sequences were selected for each category. Gene ontology of putative targets of (<b>A</b>) known miRNAs and (<b>B</b>) novel miRNAs.</p

Tissue-specific expression profiling of miRNAs identified in wheat small RNA libraries by qPCR method.

<p>(<b>A</b>) known miRNAs, (<b>B</b>) true novel miRNAs, (<b>C</b>) miRNA* sequences. PolyA tailing of total RNAs followed by cDNA synthesis and Taqman-based qPCR was employed for validation of miRNAs and normalization was carried out with wheat 5 S rRNA. Error bars represent standard error of three independent biological replicates.</p

List of identified true novel miRNAs, their star sequence and predicted targets in wheat.

<p>Accession numbers of predicted target are presented along with the possible mode of action of miRNA on the target. miRNA*: star strand of miRNA; C: mRNA cleavage; T: translational repression; NA: not applicable as no target could be predicted.</p

Summary of mapping of wheat sRNA reads in related monocot species.

<p>Putative wheat miRNAs were mapped onto available genome sequence of other monocot plants (<i>Brachypodium distachyon</i>, <i>Hordeum vulgare</i> and <i>Oryza sativa</i>) and progenitors of wheat (<i>Triticum urartu</i>, <i>Aegilops speltoides</i> and <i>Aegilops tauschii</i>). Novel miRNAs for which corresponding star sequences were found (true novel); not found (candidate novel).</p