miR390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth

Abstract

Plants adapt to different environmental conditions by constantly forming new organs in response to morphogenetic signals. Lateral roots branch from the main root in response to local auxin maxima. How a local auxin maximum translates into a robust pattern of gene activation ensuring the proper growth of the newly formed lateral root is largely unknown. Here, we demonstrate that miR390, TAS3-derived trans-acting short-interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORS (ARFs) form an auxin-responsive regulatory network controlling lateral root growth. Spatial expression analysis using reporter gene fusions, tasi/miRNA sensors, and mutant analysis showed that miR390 is specifically expressed at the sites of lateral root initiation where it triggers the biogenesis of tasiRNAs. These tasiRNAs inhibit ARF2, ARF3, and ARF4, thus releasing repression of lateral root growth. In addition, ARF2, ARF3, and ARF4 affect auxin-induced miR390 accumulation. Positive and negative feedback regulation of miR390 by ARF2, ARF3, and ARF4 thus ensures the proper definition of the miR390 expression pattern. This regulatory network maintains ARF expression in a concentration range optimal for specifying the timing of lateral root growth, a function similar to its activity during leaf development. These results also show how small regulatory RNAs integrate with auxin signaling to quantitatively regulate organ growth during development.

Figure 1.

Altered Levels of TAS3a Modify Root Architecture. (A) Schematic representation of the TAS3 pathway. miR390-loaded AGO7 cleaves the TAS3 precursor RNA. The cleavage product is converted into a double-stranded RNA by RDR6 and SGS3 and then diced into tasiARFs by DCL4 and DRB4. tasiARFs inhibit ARF2, ARF3, and ARF4 mRNA expression. (B) Root architecture of 10-d-old seedlings of the wild type (Columbia [Col]), an overexpression line (35S:TAS3a), the GABI 626B09 activation tagging line (Act. tagged), and the GABI 621G08 mutant (tas3a-1; Adenot et al., 2006). Bars = 10 mm. (C) Measurement of the average lateral root (LR) length in the different genotypes. Distribution of the population (n > 22) is represented by box plots. Differences with the wild type are indicated (***, P < 0.001; *, P < 0.05; Student's t test). (D) Numbers of lateral root primordia at specific developmental stages in 8-d-old wild-type, tas3a-1, and activation-tagged TAS3a roots (expressed as stages 1 to 7, according to [Malamy and Benfey, 1997]; mean ± se, n = 12 for each group of seedlings).

Figure 2.

Modulation of tasiARF Abundance Correlates with Lateral Root Length. (A) RNA gel blot analysis of 15μg of leaves RNA of tas3a-1 mutant (GABI 621G08), wild-type, and activation-tagged (GABI 629B09) lines. The blot was hybridized with probes complementary to TAS3 tasiRNA (Gasciolli et al., 2005), and U6 snRNA served as a loading control. (B) Schematic representation of the MIR390a locus. Position of the transcription initiation start identified by 5′ RACE is indicated by the arrow, the mature miR390 is indicated on the stem loop by a gray arrow, and the miR390* is indicated by a thick line. Position of the WiscDs insertion 30 bp upstream of the +1 is indicated. (C) RNA gel blot analysis of 15 μg of leaves or root RNA from 10-d-old wild-type (Col) or mir390a plants hybridized with miR390 or tasiARFs. U6 snRNA served as a loading control, and numbers are the ratios of miR390 to U6 signal. This experiment was done twice with similar results. (D) and (E) Measurement of the average lateral root (LR) length in the indicated genotypes. (D) The average lateral root length is reduced in homozygous mir390a plants compared with heterozygous (mir390a/+) plants. (E) The average lateral root length is reduced in dcl4-1 and rdr6 mutants compared with wild-type controls. Distribution of the populations (n > 18) is represented by box plots. **, P < 0.01; Student's t test.

Figure 3.

The Localized Expression of miR390 Governs Local tasiARF Production in Incipient Lateral Roots. (A) to (D’) Expression of reporters for TAS3a (A), MIR390a (B), an ARF3-based tasiARF sensor, and its control ([C] and [D]; Fahlgren et al., 2006) was observed in lateral root primordia either on intact 10-d-old lateral root primordia ([A] to [D]) or on transverse sections ([A’] to [D’]). The dashed lines in (A) to (D) indicate the position of transverse sections shown in (A’) to (D’). Pericycle (p), xylem (x), and phloem (ph) poles are indicated. Arrowheads in (A’) indicate pTAS3a:GUS expression in the phloem poles and parenchyma cells of the central cylinder. The arrowhead in (C’) indicates tasiARF activity in the center of the primordium. Bars = 75 μm. (E) Schematic representation of the miR390 sensor constructs. In the wild type (WT sensor), which is sensitive to miR390 action, a wild-type miR390 binding site from TAS3a (gray line) is cloned downstream of GFP, whereas in the mutated version (Mut. sensor), the miR390 binding site contains five mismatches. (F) and (G) Expression of the wild-type and mutated miR390 sensor in stage 4 lateral root primordia of 10-d-old plants. The dashed lines indicate the contour of the primordia on the confocal section and the transmitted light images (insets). Bars = 30 μm.

Figure 4.

miR390 Expression during Early Stages of Lateral Root Formation. (A) to (D) Confocal observation of pMIR390a:GUS-GFP reporter during early stages of lateral root development (Malamy and Benfey, 1997). GFP signal is in green, nuclei are stained by DAPI (blue), and the position of the xylem is marked by a dashed line. The inset in (D) is a view of a primordium from the top. (E) to (H) Confocal observation of pMIR390a:GUS-GFP and pDR5rev:erRFP reporters during early stages of lateral root development. GFP signal is in green, red fluorescent protein (RFP) is in red, and yellow indicates area of overlapping signals. The closed arrowhead in (E) indicates expression of the GFP reporter in the dividing pericycle cells, whereas the open arrowhead is expression in the xylem mesenchymal cells. (I) and (J) Observation of pMIR390a:GUS-GFP reporter before pericyle division. p, pericycle; e, endodermis; cx, cortex; ep, epidermis. (I) Confocal section showing expression of the GFP reporter in the xylem mesenchymal cells (open arrowhead). (J) Transverse section showing expression of the GUS reporter in the xylem mesenchymal cells (open arrowhead). Bars = 30 μm.

Figure 5.

miR390 Expression Responds to Auxin during Lateral Root Induction. (A) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type plants during a time course of 10 μM auxin (IAA) treatment after 24 h of 10 μM NPA pretreatment. The blot was successively probed with DNA complementary to miR390, miR156, and miR160. U6 snRNA served as a loading control. (B) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type plants. Plants were pretreated with 10 μM NPA and then for another 24 h with either DMSO (–), 10 μM cycloheximide (CHX), 10 μM IAA, or both (CHX+IAA). U6 snRNA served as a loading control. (C) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type or mir390a plants treated (+) or untreated (−) for 24 h with 10 μM IAA after 24 h of 10 μM NPA pretreatment. In (A) to (C), numbers are the ratios of miR390 to U6 signal. These experiments were done twice with similar results. (D) RT-PCR analysis of root RNA from 10-d-old wild-type plants treated for 24 h with 10 μM IAA (+) or untreated (−) after NPA pretreatment. Primers amplify specifically the MIR390a precursor and ACTIN2 (loading control) from cDNA (RT+) but not from genomic DNA (RT−). (E) and (F) Confocal analysis of pMIR390a:GUS-GFP expression in 10-d-old wild-type plants treated (+IAA) or untreated (Control) for 10 h with 10 μM IAA after NPA pretreatment. GFP is in green, and cell walls are stained by propidium iodide in red. Bars = 50 μm.

Figure 6.

miR390 Expression Depends on Signals from the Developing Lateral Root Primordium. (A) to (D) Visualization of pMIR390a:GUS-GFP activity in 10-d-old wild-type or slr mutant plants treated with 10 μM IAA ([B] and [D]) or untreated ([A] and [C]) after 24 h of NPA pretreatment. GUS assay development times were equal for (A) to (D). (E) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type or slr plants treated with 10 μM IAA for 24 h (+) or untreated (−) after NPA pretreatment. U6 snRNA served as a loading control, and numbers are the ratios of miR390 to U6 signal. This experiment was done twice with similar results.

Figure 7.

ARF2/ARF3/ARF4 Control Lateral Root Growth, and ARF4 Is Required for miR390 Expression. (A) Measurement of average lateral root length in 10-d-old seedlings expressing either the 35S:aMIR-ARFs (aMIR-ARFs) or the empty vector (Vector). Distribution of the population (n > 9) is represented by box plots. aMIR-ARFs plants have longer lateral roots than the vector controls (*, P < 0.05; Student's t test). (B) RNA gel blot analysis of 15 μg of root RNA from 10-d-old plants expressing either 35S:aMIR-ARFs or the empty vector. The plants were treated (+) with 10 μM IAA or untreated (−) after 24 h of 10μM NPA pretreatment. (C) RNA gel blot analysis of 15 μg of roots RNA from plants of the indicated genotype. (D) RNA gel blot analysis of 15 μg of root RNAs from 10-d-old plants expressing either ARF3:ARF3:GUS or its tasiARF-resistant version. The plants were treated as in (B). In (B) to (D), U6 snRNA served as a loading control, and numbers are the ratios of probe to U6 signal. These experiments were done twice with similar results. (E) to (H) Confocal observation of the pARF4:nls-3xGFP reporter construct during early stages of lateral root development. Arrowheads in (E) indicate lateral root primordia, and the inset shows transmitted light image of the same field. Bars = 20 μm. (I) Quantitative RT-PCR analysis of ARF4 transcripts in the wild type (black) and tas3a-1 mutants (red) during a time course of 10 μM auxin (IAA) treatment after 24 h of 10 μM NPA pretreatment. Values, expressed in arbitrary units (a.u.), are averages of two replicates, and error bars represent se. (J) miR390 abundance in wild-type and arf4-2 plants during a time course of 10 μM auxin (IAA) treatment after 24 h of 10 μM NPA pretreatment. Quantification of the miR390 signal was performed as in Figure 5A. (K) and (L) Confocal observation of the pMIR390a:GUS-GFP reporter in a stage 3 primordium expressed in wild-type (K) or arf4-2 mutant backgrounds (L). The arrowheads in (K) and (L) indicate the central zone of the primordium, whereas transmitted light picture of the same regions are shown in (K’) and (L’). Bars = 30 μm.

Figure 8.

A Model for the Role of the miR390/tasiARF/ARF Module during Lateral Root Growth. The diagram illustrates the spatial expression patterns of TAS3a, miR390, and ARF4 in a lateral root primordium. Hatched regions indicate the territories of overlapping gene expression. The cell layers of the primary root are indicated (x, xylem; p, pericycle; e, endodermis; cx, cortex; ep, epidermis). TAS3a accumulates in the vasculature, miR390 in the xylem, and the pericycle and the primordium in the base and flanks. The positive feedback of ARFs on miR390 supports a homeostatic model in which miR390 and ARF abundance are tightly regulated, whereas the mutual repression of miR390 and ARF4 helps to reinforce the miR390 expression pattern by removing it from the center of the primordium. Dashed arrows indicate indirect relationships.