Molecular Manipulation of the miR160/ AUXIN RESPONSE FACTOR Expression Module Impacts Root Development in Arabidopsis thaliana

Abstract

In Arabidopsis thaliana (Arabidopsis), microRNA160 (miR160) regulates the expression of AUXIN RESPONSE FACTOR10 (ARF10), ARF16 and ARF17 throughout development, including the development of the root system. We have previously shown that in addition to DOUBLE-STRANDED RNA BINDING1 (DRB1), DRB2 is also involved in controlling the rate of production of specific miRNA cohorts in the tissues where DRB2 is expressed in wild-type Arabidopsis plants. In this study, a miR160 overexpression transgene (MIR160B) and miR160-resistant transgene versions of ARF10 and ARF16 (mARF10 and mARF16) were introduced into wild-type Arabidopsis plants and the drb1 and drb2 single mutants to determine the degree of requirement of DRB2 to regulate the miR160 expression module as part of root development. Via this molecular modification approach, we show that in addition to DRB1, DRB2 is required to regulate the level of miR160 production from its precursor transcripts in Arabidopsis roots. Furthermore, we go on to correlate the altered abundance of miR160 or its ARF10, ARF16 and ARF17 target genes in the generated series of transformant lines with the enhanced development of the root system displayed by these plant lines. More specifically, promotion of primary root elongation likely stemmed from enhancement of miR160-directed ARF17 expression repression, while the promotion of lateral and adventitious root formation was the result of an elevated degree of miR160-directed regulation of ARF17 expression, and to a lesser degree, ARF10 and ARF16 expression. Taken together, the results presented in this study identify the requirement of the functional interplay between DRB1 and DRB2 to tightly control the rate of miR160 production, to in turn ensure the appropriate degree of miR160-directed ARF10, ARF16 and ARF17 gene expression regulation as part of normal root system development in Arabidopsis.

Keywords: ARF10; ARF16; ARF17; AUXIN RESPONSE FACTOR (ARF); Arabidopsis thaliana (Arabidopsis); miR160; miR160 expression module; miR160-directed gene expression regulation; microRNA (miRNA); molecular manipulation; root development.

Figure 1

Phenotypic analysis of root system architecture of 3-week-old Col-0, drb1 and drb2 plants and molecular assessment of the miR160/ARF10/ARF16/ARF17 expression module. (A) Root system development of 3-week-old Col-0, drb1 and drb2 plants. Scale bar = 1.0 cm. Quantification of primary root length (B), lateral root number (C) and adventitious root number (D) in 3-week-old Col-0, drb1 and drb2 plants. (E–M) RT-qPCR quantification of the level of expression of PRE-MIR160A (E), PRE-MIR160B (F), PRE-MIR160C (G), miR160 (H), eTM160-1 (I), eTM160-2 (J), ARF10 (K), ARF16 (L) and ARF17 (M) in the roots of 3-week-old Col-0, drb1 and drb2 plants. (E–M) Fold changes were determined by the 2^−∆∆CT method with the use of three biological replicates which contained six pooled plants per replicate. Averages of expression are represented as a fold change for each assessed transcript and were compared to the values obtained for Col-0 plants by a standard two-tailed t-test. Error bars represent the standard error of the mean (SEM) and asterisks show * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 2

Phenotypic and molecular assessment of the miR160 expression module in Col-0 plants and the Col-0/MIR160B, Col-0/mARF10, and Col-0/mARF16 transformant lines. (A) Typical root system architecture displayed by 3-week-old Col-0 plants and the Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 transformant lines. Scale bar = 1.0 cm. Quantification of primary root length (B), lateral root number (C), and adventitious root number (D) in 3-week-old Col-0, Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 plants. (E–J) RT-qPCR quantification of the level of expression of PRE-MIR160B (E), miR160 (F), eTM160-1 (G), eTM160-2 (H), ARF10 (I), and ARF16 (J) in the roots of 3-week-old Col-0, Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 plants. (E–J) Fold changes were determined by the 2^−∆∆CT method with the use of three biological replicates which contained six pooled plants per replicate. Averages of expression are represented as a fold change for each assessed transcript and were compared to the values obtained for Col-0 plants by a standard two-tailed t-test. Error bars represent the standard error of the mean (SEM) and asterisks show * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

Phenotypic and molecular assessment of the miR160 expression module in the drb1 single mutant and the drb1/MIR160B, drb1/mARF10, and drb1/mARF16 transformant lines. (A) Typical root system architecture displayed by 3-week-old drb1 plants and the drb1/MIR160B, drb1/mARF10 and drb1/mARF16 transformant lines. Scale bar = 1.0 cm. Quantification of primary root length (B), lateral root number (C), and adventitious root number (D) in 3-week-old drb1, drb1/MIR160B, drb1/mARF10 and drb1/mARF16 plants. RT-qPCR quantification of the level of expression of PRE-MIR160B (E), miR160 (F), eTM160-1 (G), eTM160-2 (H), ARF10 (I), and ARF16 (J) in the roots of 3-week-old drb1, drb1/MIR160B, drb1/mARF10 and drb1/mARF16 plants. (E–J) Fold changes were determined by the 2^−∆∆CT method with the use of three biological replicates which contained six pooled plants per replicate. Averages of expression are represented as a fold change for each assessed transcript and were compared to the values obtained for drb1 plants by a standard two-tailed t-test. Error bars represent the standard error of the mean (SEM) and asterisks show * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 4

Phenotypic and molecular assessment of the miR160 expression module in the drb2 single mutant and the drb2/MIR160B, drb2/mARF10, and drb2/mARF16 transformant lines. (A) Typical root system architecture displayed by 3-week-old drb2 plants and the drb2/MIR160B, drb2/mARF10 and drb2/mARF16 transformant lines. Scale bar = 1.0 cm. Quantification of primary root length (B), lateral root number (C), and adventitious root number (D) in 3-week-old drb2, drb2/MIR160B, drb2/mARF10 and drb2/mARF16 plants. (E–J) RT-qPCR quantification of transcript abundance of PRE-MIR160B (E), miR160 (F), eTM160-1 (G), eTM160-2 (H), ARF10 (I), and ARF16 (J) in the roots of 3-week-old drb2, drb2/MIR160B, drb2/mARF10 and drb2/mARF16 plants. (E–J) Fold changes were determined by the 2^−∆∆CT method with the use of three biological replicates which contained six pooled plants per replicate. Averages of expression are represented as a fold change for each assessed transcript and were compared to the values obtained for drb2 plants by a standard two-tailed t-test. Error bars represent the standard error of the mean (SEM) and asterisks show * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.