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

Abstract

Oryza coarctata, 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 O. coarctata were sequenced, which yielded 338 known and 95 novel miRNAs. Additionally, we used publicly available transcriptomics data of O. coarctata which led to the discovery of additional 48 conserved miRNAs along with their pre-miRNA sequences through in silico 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 O. coarctata.

Fig 1. Adult saplings of Oryza coarcta growing under green house (left), leaf sample from two-month old sapling taken for preparation of small RNA seq library (right).

Fig 2. Number and expression of miRNAs of O. coarctata.

A) Number of miRNAs in O. coarctata. 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.

Fig 3. Example of predicted secondary structure of some miRNAs.

A) Novel microRNAs of O. coarctata. B) Secondary structure of some miRNAs as detected by in silico analysis.

Fig 4. Locations and nature of novel miRNAs of O coarctata.

A) Chromosomal distribution of miRNAs. B) Within chromosome locations of different novel miRNAs.

Fig 5. Scatter plot of novel miRNA expression level.

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.

Fig 6. Mapping of target mRNA cleavage sites of miR393a by 5’ RACE.

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.

Fig 7. Gene ontology (GO) categories of target genes of known and new miRNA families.

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

Fig 8. qRT-PCR analysis of the relative expression of miRNAs and targets.

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.

Fig 9. Predicted miRNA-target interaction in O. coarctata.

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