DRB1, DRB2 and DRB4 Are Required for an Appropriate miRNA-Mediated Molecular Response to Osmotic Stress in Arabidopsis thaliana

Abstract

Arabidopsis thaliana (Arabidopsis) double-stranded RNA binding (DRB) proteins DRB1, DRB2 and DRB4 perform essential roles in microRNA (miRNA) production, with many of the produced miRNAs mediating aspects of the molecular response of Arabidopsis to abiotic stress. Exposure of the drb1, drb2 and drb4 mutants to mannitol stress showed drb2 to be the most sensitive to this form of osmotic stress. Profiling of the miRNA landscapes of mannitol-stressed drb1, drb2 and drb4 seedlings via small RNA sequencing, and comparison of these to the profile of mannitol-stressed wild-type Arabidopsis plants, revealed that the ability of the drb1 and drb2 mutants to mount an appropriate miRNA-mediated molecular response to mannitol stress was defective. RT-qPCR was next used to further characterize seven miRNA/target gene expression modules, with this analysis identifying DRB1 as the primary DRB protein required for miR160, miR164, miR167 and miR396 production. In addition, via its antagonism of DRB1 function, DRB2 was shown by RT-qPCR to play a secondary role in regulating the production of these four miRNAs. This analysis further showed that DRB1, DRB2 and DRB4 are all required to regulate the production of miR399 and miR408, and that DRB4 is the primary DRB protein required to produce the non-conserved miRNA, miR858. Finally, RT-qPCR was used to reveal that each of the seven characterized miRNA/target gene expression modules responded differently to mannitol-induced osmotic stress in each of the four assessed Arabidopsis lines. In summary, this research has identified mannitol-stress-responsive miRNA/target gene expression modules that can be molecularly manipulated in the future to generate novel Arabidopsis lines with increased tolerance to this form of osmotic stress.