Number and expression of miRNAs of <i>O</i>. <i>coarctata</i>.

<p>A) Number of miRNAs in <i>O</i>. <i>coarctata</i>. X = Control Y = 24h salt-treated. The first numerical figure indicates known and second numerical figure indicate novel miRNAs. B) Expressions of miRNA that are found in both the libraries.</p

Identification of Novel and Conserved miRNAs from Extreme Halophyte, <i>Oryza coarctata</i>, a Wild Relative of Rice

<div><p><i>Oryza coarctata</i>, a halophyte and wild relative of rice, is grown normally in saline water. MicroRNAs (miRNAs) are non-coding RNAs that play pivotal roles in every domain of life including stress response. There are very few reports on the discovery of salt-responsive miRNAs from halophytes. In this study, two small RNA libraries, one each from the control and salt-treated (450 mM NaCl for 24 h) leaves of <i>O</i>. <i>coarctata</i> were sequenced, which yielded 338 known and 95 novel miRNAs. Additionally, we used publicly available transcriptomics data of <i>O</i>. <i>coarctata</i> which led to the discovery of additional 48 conserved miRNAs along with their pre-miRNA sequences through <i>in silico</i> analysis. In total, 36 known and 7 novel miRNAs were up-regulated whereas, 12 known and 7 novel miRNAs were down-regulated under salinity stress. Further, 233 and 154 target genes were predicted for 48 known and 14 novel differentially regulated miRNAs respectively. These targets with the help of gene ontology analysis were found to be involved in several important biological processes that could be involved in salinity tolerance. Relative expression trends of majority of the miRNAs as detected by real time-PCR as well as predicted by Illumina sequencing were found to be coherent. Additionally, expression of most of the target genes was negatively correlated with their corresponding miRNAs. Thus, the present study provides an account of miRNA-target networking that is involved in salinity adaption of <i>O</i>. <i>coarctata</i>.</p></div

Gene ontology (GO) categories of target genes of known and new miRNA families.

<p>Categorization of miRNA target genes was performed according to the three GO domains; biological process, cellular component and molecular function.</p

Scatter plot of novel miRNA expression level.

<p>Expression levels are normalized to TPM. Data points lower or upper the slope line represent down- or up-regulated miRNAs in panel. The changes in up- and down-regulated miRNAs are greater than 1 fold.</p

Locations and nature of novel miRNAs of <i>O coarctata</i>.

<p>A) Chromosomal distribution of miRNAs. B) Within chromosome locations of different novel miRNAs.</p

Predicted miRNA-target interaction in <i>O</i>. <i>coarctata</i>.

<p>Target genes are denoted by italics: ubiquitin-conjugating enzyme(<i>UBC</i>); Sucrose transporter (<i>ST</i>); Major facilitor super family protein (<i>MFS</i>); MYB; APETALA2 (<i>AP2</i>); SPB-like proteins(<i>SPL</i>); auxin response factor(<i>ARF</i>); nuclear transcription factor Y (<i>NF-Y</i>); NAC domain-containing proteins (<i>NAC</i>); ATP synthatase (<i>ATPs</i>); peroxidase (<i>POX</i>); Anthocyanidin synthatase (<i>ACS</i>); 4-coumarate-CoA liagase-1 (<i>CML</i>); Ubiquitin protein liagase (<i>UBL</i>); Ring Finger protein (<i>RFP</i>); Cullin-1(<i>CUL</i>); serine/threonine-protein kinase 38 (<i>STK</i>); Calcium binding protein (<i>CBP</i>); Cation transporter HKT7 (<i>HKT7</i>); Calmodulin (<i>CAM</i>).</p

Sensitive Valence Structures of [(pap)₂Ru(Q)]^<i>n</i> (<i>n</i> = +2, +1, 0, −1, −2) with Two Different Redox Noninnocent Ligands, Q = 3,5-Di-<i>tert</i>-butyl-<i>N</i>-aryl-1,2-benzoquinonemonoimine and pap = 2-Phenylazopyridine

The complexes [(pap)2Ru(Q)]ClO4, [1]ClO4−[4]ClO4, with two different redox noninnocent ligands, Q = 3,5-di-tert-butyl-N-aryl-1,2-benzoquinonemonoimine (-aryl = m-(Cl)2C6H3 (1+), C6H5 (2+), m-(OCH3)2C6H3 (3+), and m-(tBu)2C6H3 (4+)) and pap = 2-phenylazopyridine, have been synthesized and characterized using various analytical techniques. The single-crystal X-ray structure of the representative [2]ClO4·C7H8 exhibits multiple intermolecular C−H···O hydrogen bondings and C−H···π interactions. The C1−O1 = 1.287(4) (density functional theory, DFT, 1.311) and C6−N1 = 1.320(4) (DFT, 1.353) Å and intraring bond distances associated with the sensitive quinine (Q) moiety along with the azo(pap) bond distances, N3−N4 = 1.278(4) (DFT, 1.297) and N6−N7 = 1.271(4) (DFT, 1.289) Å, in 2+ justify the [(pap)2RuII(Q•−)]+ valence configuration at the native state of 1+−4+. Consequently, Mulliken spin densities on Q, pap, and Ru in 2+ are calculated to be 0.8636, 0.1040, and 0.0187, respectively, and 1+−4+ exhibit free radical sharp EPR spectra and one weak and broad transition around 1000 nm in CH3CN due to interligand transition involving a singly occupied molecular orbital (SOMO) of Q•− and the vacant π* orbital of pap. Compounds 1+−4+ undergo a quasi-reversible oxidation and three successive reductions. The valence structure of the electron paramagnetic resonance (EPR)-inactive oxidized state in 12+−42+ has been established as [(pap)2RuII(Q°)]2+ instead of the alternate formalism of antiferromagnetically coupled [(pap)2RuIII(Q•−)]2+ on the basis of the DFT calculations on the optimized 2+, which predict that the singly occupied molecular orbital is primarily composed of Q with 77% contribution. Accordingly, the optimized structure of 22+ predicts shorter C1−O1 (1.264) and C6−N1 (1.317 Å) distances and longer Ru1−O1 (2.080) and Ru1−N1 (2.088 Å) distances. Compounds 12+−42+ exhibit the lowest energy transitions around 600 nm, corresponding to Ru(dπ)/Q(π) → pap(π*). The presence of two sets of strongly π-acceptor ligands, pap and Q, in 12+−42+ stabilizes the Ru(II) state to a large extent such that the further oxidation of {RuII−Q°} → {RuIII−Q°} has not been detected within +2.0 V versus a saturated calomel electrode. The EPR-inactive reduced states 1−4 have been formulated as [(pap)2RuII(Q2−)] over the antiferromagnetically coupled alternate configuration, [(pap)(pap•−)RuII(Q•−)]. The optimized structure of 2 predicts sensitive C1−O1 and C6−N1 bond distances of 1.337 and 1.390 Å, respectively, close to the doubly reduced Q2− state, whereas the NN distances of pap, N3−N4 = 1.299 and N6−N7 = 1.306 Å, remain close to the neutral state. In corroboration with the doubly reduced Q2− state, 1−4 exhibit a moderately strong interligand π(Q2−) → π*(pap) transition in the near-IR region near 1300 nm. The subsequent two reductions are naturally centered around the azo functions of the pap ligands

Mapping of target mRNA cleavage sites of miR393a by 5’ RACE.

<p>The target of miR393a (TIR1) encodes an auxin receptor family protein. The arrow indicates the cleavage site, and the numbers above the arrow denote the frequencies of the sequenced clones.</p

qRT-PCR analysis of the relative expression of miRNAs and targets.

<p>A) Comparison of miRNAs (fold changes) between illumina reads and qRT-PCR between control and salt-treated library. B) Relative expression of miRNAs and their targets under control and salinity stress condition. The data represents the mean values ± SD of three replicates.</p

Example of predicted secondary structure of some miRNAs.

<p>A) Novel microRNAs of <i>O</i>. <i>coarctata</i>. B) Secondary structure of some miRNAs as detected by <i>in silico</i> analysis.</p