Nitrate-responsive miR393/AFB3 regulatory module controls root system architecture in Arabidopsis thaliana

Abstract

One of the most striking examples of plant developmental plasticity to changing environmental conditions is the modulation of root system architecture (RSA) in response to nitrate supply. Despite the fundamental and applied significance of understanding this process, the molecular mechanisms behind nitrate-regulated changes in developmental programs are still largely unknown. Small RNAs (sRNAs) have emerged as master regulators of gene expression in plants and other organisms. To evaluate the role of sRNAs in the nitrate response, we sequenced sRNAs from control and nitrate-treated Arabidopsis seedlings using the 454 sequencing technology. miR393 was induced by nitrate in these experiments. miR393 targets transcripts that code for a basic helix-loop-helix (bHLH) transcription factor and for the auxin receptors TIR1, AFB1, AFB2, and AFB3. However, only AFB3 was regulated by nitrate in roots under our experimental conditions. Analysis of the expression of this miR393/AFB3 module, revealed an incoherent feed-forward mechanism that is induced by nitrate and repressed by N metabolites generated by nitrate reduction and assimilation. To understand the functional role of this N-regulatory module for plant development, we analyzed the RSA response to nitrate in AFB3 insertional mutant plants and in miR393 overexpressors. RSA analysis in these plants revealed that both primary and lateral root growth responses to nitrate were altered. Interestingly, regulation of RSA by nitrate was specifically mediated by AFB3, indicating that miR393/AFB3 is a unique N-responsive module that controls root system architecture in response to external and internal N availability in Arabidopsis.

Fig. 1.

Nitrate consistently regulates miR393 and its target AFB3 in Arabidopsis roots. Plants were grown hydroponically for 14 days with ammonium as the sole N source and were treated with 5 mM KNO₃ or 5 mM KCl for the times indicated. Root transcript levels for bHLH77, AFB1, AFB2, AFB3, and mature miR393 were analyzed by real-time qPCR. We show the mean and standard error for three biological replicates. The asterisk indicates means that significantly differ between the control and treatment conditions (P < 0.01).

Fig. 2.

AFB3 transcript is cleaved by miR393 in response to nitrate. (A) Wild-type Col-0 plants and miR393 overexpressor plants (28) were grown in 0.5× MS salts supplemented with 30 mM sucrose in Petri dishes for 14 days. AFB3 levels were analyzed in seedlings using qPCR. We show the mean and standard error for three biological replicates. (B) Plants were grown hydroponically for 14 days with ammonium as the sole N source and were treated with 5 mM KNO₃ for 2 h. Poly(A)⁺ RNA was extracted from roots and a modified RLM-RACE procedure was used to amplify a miR393 cleavage product from AFB3 (28). NTC, no template control. (C) pAFB3:mAFB3-GUS plants (33) were grown as described in B and were treated with 5 mM KNO₃ for the times indicated. AFB3 transcript levels in roots were analyzed by real-time qPCR. Values are presented as the log₂ ratio between the treatment level and the time 0 levels. As a reference, we also present the AFB3 transcript levels in wild-type plants from Fig. 1.

Fig. 3.

AFB3 is directly induced by nitrate and is under posttranscriptional regulation by N metabolites produced after nitrate reduction by a pathway involving miR393. Nitrate reductase-null mutant plants (34) were grown hydroponically as described before and were treated with 5 mM KNO₃ or 5 mM KCl for the times indicated. AFB3 transcript levels and mature miR393 levels were analyzed by real-time qPCR in roots. We show the mean and standard error for three biological replicates. The asterisk indicates means that significantly differ between control and treatment conditions (P < 0.01).

Fig. 4.

Nitrate regulates primary root growth by a pathway involving AFB3. Plants were grown hydroponically as described before and were treated for the times indicated. (A) pAFB3::GUS plants (35) were treated for 1 h with 5 mM KNO₃ or KCl and were then stained for GUS activity for 4 h. Qualitative GUS staining was analyzed using DIC optics. Photographs are representative of at least 15 stained plants. (Scale bar, 100 μm.) (B) Wild-type plants were treated for 1 h with 5 mM KNO₃ or KCl. Root tips were excised from nitrate-treated or control-treated plants and AFB3 RNA levels were measured using qPCR. Bars represent SE. The asterisk represents means that significantly differ (P < 0.01). (C) Auxin reporter DR5::GUS plants (36) were treated for 1 h with 5 mM KNO₃ or KCl and were then stained for GUS activity for 12 h. Qualitative GUS staining was analyzed using DIC optics. Photographs are representative of at least 15 stained plants. (Scale bar, 100 μm.) (D) Primary root length of Ws wild-type plants or afb3-1 mutant plants was measured using the ImageJ program after 3 days of 5 mM KNO₃ or KCl treatment. Bars represent standard errors. Different letters represent significantly different means (P < 0.01). (E) Primary root length of Col-0 wild-type plants or 35S::miR393 overexpressor plants (28) was measured using the ImageJ program after 3 days of 5-mM KNO₃ or KCl treatment. Bars represent standard errors. Different letters represent statistically different means (P < 0.01).

Fig. 5.

Nitrate regulates lateral root growth by a pathway mediated by AFB3. Plants were grown hydroponically as described before and were treated for the times indicated. (A) pAFB3::GUS plants (35) were treated for 1 h with 5 mM KNO₃ or KCl and were then stained for GUS activity for 4 h. Qualitative GUS staining was analyzed using DIC optics. Photographs are representative of at least 15 stained plants. (Scale bar, 100 μm.) (B) Pericycle marker line plants were treated for 1.5 h with 5 mM KNO₃ or KCl. Protoplast were prepared from roots and pericycle cells expressing GFP were sorted by FACS. RNA levels for AFB3 were measured using qPCR. Bars represent standard errors. The asterisk represents statistically different means (P < 0.05). (C) The number of initiating and emerging lateral roots of afb3-1 mutants (35) or Ws wild-type plants treated for 3 days with 5 mM KNO₃ or KCl was counted using DIC optics. Bars represent standard errors. Different letters indicate statistically different means (P < 0.01). (D) The number of initiating and emerging lateral roots of 35S::miR393 overexpressor plants (28) or Col-0 wild-type plants treated for 3 days with 5 mM KNO₃ or KCl was counted using DIC optics. Bars represent SE. Different letters indicate statistically different means (P < 0.01).