Identification and characterization of microRNAs responsive to fluoride toxicity in rice (Oryza sativa L.)

MicroRNAs (miRNAs) are a class of small non-coding RNAs that act as important modulators of gene expression related to several stress responses in plants. While several miRNAs have been implicated in the modulation of multiple abiotic and biotic stresses in rice, there role in response to fluoride stress is yet to be explored. In the present study, fourteen conserved rice miRNAs with proven role in multiple stress response were analysed to identify differentially expressed miRNAs in response to fluoride toxicity in two popular rice varieties- Gobindobhog (GB; F-tolerant) and IR64 (F-sensitive). Stem-Loop RT-PCR revealed that miR156, miR166, and miR171 were significantly induced in GB seedlings post treatment with fluoride. Likewise, miR160, miR319, miR396, and miR444 were prominently induced in the fluoride-sensitive IR64. Additionally, miR393 was significantly induced post-treatment with fluoride stress in both the genotypes exhibiting a basal response to fluoride toxicity. Further, we computationally predicted the miRNA targets many of which encoded transcription factors associated with stress response mechanism. The miRNA targets were experimentally validated using ligation mediated 5′ rapid amplification of cDNA ends analysis. Quantitative RT-PCR analysis of nine selected miRNA target genes (Os11g30370, Os06g47150, Os06g03670, Os04g48290, Os02g44360, Os08g34380, Os05g05800, Os04g57050, Os04g51350) revealed simultaneous reciprocal changes in the expression patterns of the miRNAs and the corresponding target genes suggesting their involvement in the modulation of fluoride stress response in rice. Analysis of proximal promoter sequences of the F-responsive miRNAs revealed that these miRNAs possess stress-responsive, elicitor and hormonal related motifs. Overall, our results suggest that multiple conserved miRNAs are involved in fluoride toxicity and a miRNA-mediated regulation of signal response is critical for rice response to fluoride stress.