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.

Keywords: Arabidopsis thaliana (Arabidopsis); DRB1; DRB2; DRB4; double-stranded RNA binding (DRB) protein; mannitol; miRNA-directed gene expression regulation; microRNA (miRNA); osmotic stress.

Figure 1

Phenotypic and physiological assessment of 15-day-old Col-0, drb1, drb2 and drb4 seedlings following a 7-day growth period on solid growth medium supplemented with 200 mM mannitol. (A) Phenotypes expressed by 15-day-old control-grown Col-0/Ns, drb1/Ns, drb2/Ns and drb4/Ns seedlings (top panel) and mannitol-stressed Col-0/Mann, drb1/Mann, drb2/Mann and drb4/Mann seedlings (bottom panels). Scale bar = 1.0 cm. (B–G) All quantified phenotypic and physiological parameters are compared to those obtained for the control-grown counterpart of each Arabidopsis line, with differences in whole-seedling fresh weight (B), rosette area (C), primarily root length (D), anthocyanin content (E) and chlorophyll a (F) and b (G) content determined via the assessment of four biological replicates which consisted of pools of 12 individual plants. (B–G) Error bars represent the standard deviation (±SD) of the four biological replicates, and the presence of an asterisk (*) above a column represents a statistically significant difference between the mannitol-stressed sample and the control sample (p-value: * < 0.05; ** < 0.005; *** < 0.001).

Figure 2

Molecular assessment via RT-qPCR of the effects of the exposure of 15-day-old Col-0, drb1, drb2 and drb4 seedlings to the osmotic stress agent mannitol. (A) RT-qPCR analysis of the expression of the P5CS1 stress response gene in 15-day-old Col-0, drb1, drb2 and drb4 seedlings following a 7-day exposure period to mannitol stress. (B–E) RT-qPCR assessment of DCL1 (B), DRB1 (C), DRB2 (D) and DRB4 (E) gene expression in 15-day-old Col-0, drb1, drb2 and drb4 seedlings following a 7-day exposure period to osmotic stress. Error bars represent the ±SD of four biological replicates, and the presence of an asterisk (*) above a column represents a statistically significant difference between the sample exposed to stress and the control sample (p-value: * < 0.05; ** < 0.005; *** < 0.001).

Figure 3

Profiling of the miRNA landscapes of 15-day-old mannitol stressed Col-0, drb1, drb2 and drb4 seedlings via small RNA sequencing. A sRNA-Seq approach was used to establish the degree of alteration to the miRNA landscapes of 15-day-old mannitol-stressed Col-0, drb1, drb2 and drb4 seedlings. For each vertical column of the heatmap, an individual tile represents a single miRNA per assessed Arabidopsis line, where the intensity of the red-colored shading indicates the extent of abundance upregulation for an individual miRNA, while the intensity of the blue-colored shading represents the extent of abundance downregulation for an individual miRNA.

Figure 4

Profiling of miRNA accumulation in 15-day-old mannitol-stressed Col-0, drb1, drb2 and drb4 seedlings by sRNA-Seq and RT-qPCR. (A) Profiling by sRNA-Seq of the abundance of all members of the MIR160, MIR164, MIR167, MIRR396, MIR399, MIR408 and MIR858 gene families in mannitol-stressed Col-0, drb1, drb2 and drb4 seedlings. The shading intensity (light to dark) of each tile of each column depicts the degree of abundance change presented as a standard fold change. (B–H) RT-qPCR quantification of miR160 (B), miR164 (C), miR167 (D), miR396 (E), miR399 (F), miR408 (G) and miR858 (H) abundance in 15-day-old Col-0, drb1, drb2 and drb4 seedlings following the 7-day osmotic stress treatment period with miRNA abundance compared to the control grown counterpart of each Arabidopsis line. Error bars represent the ±SD of four biological replicates, and each biological replicate consisted of a pool of 12 individual plants. The presence of an asterisk (*) above a column represents a statistically significant difference between the mannitol-stressed sample and the control sample (p-value: * < 0.05; ** < 0.005; *** < 0.001).

Figure 5

RT-qPCR assessment of miRNA target gene expression in 15-day-old mannitol-stressed Col-0, drb1, drb2 and drb4 seedlings. (A–G) RT-qPCR analysis of the expression of ARF17 (A), CUC1 (B), ARF8 (C), GRF7 (D), PHO2 (E), LAC3 (F) and ERF7 (G), the representative target genes of miR160, miR164, miR167, miR396, miR399, miR408 and miR858, respectively, in 15-day-old mannitol-stressed Col-0, drb1, drb2 and drb4 seedlings. Target gene expression (presented as a standard fold change) in each mannitol-stressed Arabidopsis line was determined via direct comparison to the level of target gene expression in the control grown counterpart of each line. Error bars represent the ±SD of four biological replicates, with each biological replicate consisting of a pool of 12 individual plants. The presence of an asterisk (*) above a column represents a statistically significant difference between the mannitol-stressed sample and the control sample (p-value: * < 0.05; ** < 0.005; *** < 0.001).