A Genome-Wide Perspective of miRNAome in Response to High Temperature, Salinity and Drought Stresses in <i>Brassica juncea</i> (Czern) L

<div><p>Micro RNAs (miRNAs) are involved in diverse biological processes including adaptive response towards abiotic stresses. To unravel small RNAs and more specifically miRNAs that can potentially regulate determinants of abiotic stress tolerance, next generation sequencing of <i>B. juncea</i> seedlings subjected to high temperature, high salt and drought conditions was carried out. With the help of UEA sRNA workbench software package, 51 conserved miRNAs belonging to 30 miRNA families were identified. As there was limited genomic information available for <i>B. juncea</i>, we generated and assembled its genome sequence at a low coverage. Using the generated sequence and other publically available <i>Brassica</i> genomic/transcriptomic resources as mapping reference, 126 novel (not reported in any plant species) were discovered for the first time in <i>B. juncea</i>. Further analysis also revealed existence of 32 and 37 star sequences for conserved and novel miRNAs, respectively. The expression of selected conserved and novel miRNAs under conditions of different abiotic stresses was revalidated through universal TaqMan based real time PCR. Putative targets of identified conserved and novel miRNAs were predicted in <i>B. rapa</i> to gain insights into functional roles manifested by <i>B. juncea</i> miRNAs. Furthermore, SPL2-like, ARF17-like and a NAC domain containing protein were experimentally validated as targets of miR156, miR160 and miR164 respectively. Investigation of gene ontologies linked with targets of known and novel miRNAs forecasted their involvement in various biological functions.</p></div

The miRNA sequences identified in <i>B. juncea</i> were analysed for identical matches in family <i>Brassica</i>ceae.

<p>The miRNA family, sequence and miRBase nomenclature of these identical miRNA sequences of each species is presented.</p

Filtering of small RNA reads. Purity filtered sequences were processed through an elimination pipeline.

<p>The number and percentage (in parentheses) of the sequences removed for redundant (column 2) and unique sequences (column 3) are tabulated. The category of the removed sequences is represented in column 1.</p

Cleavage site mapping of selected miRNAs using modified RLM-RACE.

<p>Targets of the <i>B. juncea</i> miR156 (Bju-miR156), miR160 (Bju-miR160) and miR164 (Bju-miR164) were predicted by psRNA target finder. Primers were designed on the basis of the predicted sequences (TC2101178, TC165518 and TC211305). Cleavage products of three miRNA targets were amplified from RACE library, cloned in pGEMT vector and sequenced. The binding region between the miRNAs and the targets is shown. The arrows indicate cleavage site of transcript and the number of positive clones out of the total clones sequenced is indicated in parentheses above the arrow.</p

Expression profiling of miRNAs and their targets.

<p>The relative abundances of miR156, miR160, miR164 and their respective targets i.e., SPL2 (TC2101178), ARF17 (TC165518) and NAC1 (TC211305) was measured under different abiotic stress conditions using quantitative real time PCR. <i>B. juncea</i> seedlings were subjected for varied durations to either high temperature stress either at 35°C (BJH-1) and 42°C (BJH-2) or salinity stress at 150 mM NaCl (BJS-1) and 250 mM NaCl (BJS-2) or drought stress using 20% PEG (BJD-1) or 300 mM mannitol (BJD-2). The mean of three independent biological replicates is presented.</p

Putative targets of selected novel miRNAs.

<p>Expression levels of few identified novel miRNAs was determined using quantitative PCR. The putative targets of these novel miRNAs were predicted in <i>B. rapa</i> using psRNAtarget software. The accession number and description of predicted targets (based on their homology with <i>A. thaliana</i>) for each novel miRNA are presented.</p

Expression profiling of conserved (A) and novel miRNAs (B) was performed to validate the predicted miRNAs.

<p>Quantitative PCR was performed using TaqMan chemistry. The relative abundance (Y-axis) was calculated using the ΔΔCt method. <i>B. juncea</i> seedlings were subjected for varied durations to either high temperature stress either at 35°C (BJH-1) and 42°C (BJH-2) or salinity stress at 150 mM NaCl (BJS-1) and 250 mM NaCl (BJS-2) or drought stress using 20% PEG (BJD-1) and 300 mM mannitol (BJD-2). The mean of three independent biological replicates is presented.</p

The digital expression values obtained through high-throughput sequencing were normalized using TPM (transcripts per million) method and used to calculate digital expression fold change [Log 2 (abiotic stress treated sample/control sample)] in high temperature treated (BJH), salinity stress treated (BJS) and drought stress treated (BJD) <i>B. juncea</i> seedlings library with respect to untreated seedlings library.

<p>Thus deduced digital expression fold change is presented as heat map for conserved (A) and novel (B) miRNA sequences.</p

Gene ontology analysis of predicted targets of conserved (A) and novel (B) miRNAs.

<p>For each GO category (molecular function, cellular component and biological process) top 5 GO terms obtained through Blast2GO resource (<a href="http://www.blast2go.com" target="_blank">www.blast2go.com</a>) are presented.</p