MicroRNA-Mediated Responses to Cadmium Stress in Arabidopsis thaliana

Abstract

In recent decades, the presence of cadmium (Cd) in the environment has increased significantly due to anthropogenic activities. Cd is taken up from the soil by plant roots for its subsequent translocation to shoots. However, Cd is a non-essential heavy metal and is therefore toxic to plants when it over-accumulates. MicroRNA (miRNA)-directed gene expression regulation is central to the response of a plant to Cd stress. Here, we document the miRNA-directed response of wild-type Arabidopsis thaliana (Arabidopsis) plants and the drb1, drb2 and drb4 mutant lines to Cd stress. Phenotypic and physiological analyses revealed the drb1 mutant to display the highest degree of tolerance to the imposed stress while the drb2 mutant was the most sensitive. RT-qPCR-based molecular profiling of miRNA abundance and miRNA target gene expression revealed DRB1 to be the primary double-stranded RNA binding (DRB) protein required for the production of six of the seven Cd-responsive miRNAs analyzed. However, DRB2, and not DRB1, was determined to be required for miR396 production. RT-qPCR further inferred that transcript cleavage was the RNA silencing mechanism directed by each assessed miRNA to control miRNA target gene expression. Taken together, the results presented here reveal the complexity of the miRNA-directed molecular response of Arabidopsis to Cd stress.

Keywords: Arabidopsis thaliana (Arabidopsis); RT-qPCR; cadmium (Cd) stress; double-stranded RNA binding (DRB) protein; miRNA-directed gene expression regulation; microRNA (miRNA).

Figure 1

The phenotypic and physiological responses of the Arabidopsis lines Col-0, drb1, drb2 and drb4 to cadmium stress. (A) The phenotypes displayed by 15-day-old control (top panels) and Cd-stressed (bottom panels) Col-0, drb1, drb2 and drb4 plants. Bar = 1.0 cm. Quantification of the phenotypic parameters, (B) rosette area (mm²), (C) primary root length (mm) and (D) whole seedling fresh weight (mg), of 15-day-old Cd-stressed Col-0, drb1, drb2 and drb4 seedlings compared to those of the control grown counterpart of each plant line. Determination of the physiological parameters, (E) total chlorophyll content (mg/g FW) and the (F) degree of cell membrane damage (presented as a percentage (%) of the absorbance of the Col-0/Ns sample at wavelength 600 nm), as determined by spectrophotometry of control and Cd-stressed Col-0, drb1, drb2 and drb4 seedlings. (B–F) Statistical data were analyzed using one-way ANOVA and Tukey’s post-hoc tests. The statistically significant differences are indicated by a different letter (p-value < 0.05) above each column of each histogram.

Figure 2

RT-qPCR quantification of the expression of Arabidopsis genes known to be responsive to cadmium stress. A standard RT-qPCR approach was used to quantify the transcript abundance of a select group of Arabidopsis genes known to be responsive to Cd stress, including (A) Squamosa promoter binding protein-like 7 (SPL7); (B) PDR8; (C) natural resistance-associated macrophage protein (NRAMP6); (D) FE superoxide dismutase 1 (FSD1); (E) heavy metal ATPase 2 (HMA2) and (F) heavy metal ATPase 4 (HMA4). The expression of these six genes was analyzed in 15-day-old Col-0, drb1, drb2 and drb4 plants following their exposure to a 7-day 50 μM CdCl₂ stress treatment regime for comparison to the control grown counterpart of each Arabidopsis line. (A–F) Statistical data were analyzed using one-way ANOVA and Tukey’s post-hoc tests. The statistically significant differences are indicated by a different letter (p-value < 0.05) above each column of each histogram.

Figure 3

Molecular profiling of the expression modules of three auxin pathway-specific microRNAs following the exposure of Col-0, drb1, drb2 and drb4 plants to cadmium stress. RT-qPCR quantification of miR160 abundance (A) and the expression of its target genes, ARF10 (B), ARF16 (C) and ARF17 (D), in 15-day-old control and Cd-stressed Col-0, drb1, drb2 and drb4 plants. RT-qPCR quantification of miR167 abundance (E) and of the expression of its ARF target genes, ARF6 (F) and ARF8 (G), in 15-day-old control and Cd-stressed Col-0, drb1, drb2 and drb4 plants. Profiling of miR393 abundance (H) and of the expression of its primary target gene, TIR1 (I), in control and Cd-stressed Col-0, drb1, drb2 and drb4 whole seedlings by RT-qPCR. (A–I) Statistical data were analyzed using one-way ANOVA and Tukey’s post-hoc tests. The statistically significant differences are indicated by a different letter (p-value < 0.05) above each column of each histogram.

Figure 4

RT-qPCR quantification of the abundance of three Arabidopsis abiotic stress responsive microRNAs and the expression of their target genes in cadmium-stressed Col-0, drb1, drb2 and drb4 seedlings. RT-qPCR assessment of miR395 abundance (A) and the expression of its target gene, ATPS1 (B), in control and Cd-stressed Col-0, drb1, drb2 and drb4 whole seedlings. Quantification of the abundance of miR399 (C) and the expression of its target gene PHO2 (D) in Col-0, drb1, drb2 and drb4 whole seedlings cultivated in either control conditions or a Cd-stress environment. RT-qPCR analysis of miR408 abundance (E) and the expression of its target gene, LAC3 (F), in 15-day-old control and Cd-stressed Col-0, drb1, drb2 and drb4 whole seedlings. (A–F) Statistical data were analyzed using one-way ANOVA and Tukey’s post-hoc tests. The statistically significant differences are indicated by a different letter (p-value < 0.05) above each column of each histogram.

Figure 5

RT-qPCR quantification of the miR396 abundance and GRF target gene expression in control and cadmium-stressed Col-0, drb1, drb2 and drb4 plants. Quantification of miR396 abundance (A) and the expression of its GRF target genes, GRF1 (B), GRF2 (C), GRF3 (D), GRF7 (E), GRF8 (F) and GRF9 (G), via RT-qPCR assessment of 15-day-old control and Cd-stressed Col-0, drb1, drb2 and drb4 whole seedlings. (A–G) Statistical data were analyzed using one-way ANOVA and Tukey’s post-hoc tests. The statistically significant differences are indicated by a different letter (p-value < 0.05) above each column of each histogram.