Modulation of plant root growth by nitrogen source‐defined regulation of polar auxin transport

Abstract Availability of the essential macronutrient nitrogen in soil plays a critical role in plant growth, development, and impacts agricultural productivity. Plants have evolved different strategies for sensing and responding to heterogeneous nitrogen distribution. Modulation of root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to distinct nitrogen sources, such as nitrate or ammonium, are poorly understood. Here, we show that growth as manifested by cell division and elongation is synchronized by coordinated auxin flux between two adjacent outer tissue layers of the root. This coordination is achieved by nitrate‐dependent dephosphorylation of the PIN2 auxin efflux carrier at a previously uncharacterized phosphorylation site, leading to subsequent PIN2 lateralization and thereby regulating auxin flow between adjacent tissues. A dynamic computer model based on our experimental data successfully recapitulates experimental observations. Our study provides mechanistic insights broadening our understanding of root growth mechanisms in dynamic environments.

. Monitoring basipetal auxin transport and auxin response at root tips of Col-0 and eir1-4 roots transferred to ammonium or nitrate-containing medium.
A Basipetal (shootward) auxin transport measurements in Col-0 and eir1-4 roots grown on control Murashige and Skoog (MS) or with either nitrate or ammonium supplied media. 3 H-IAA was applied at the root tip of 7 DAG wild-type (Col-0) or eir1-4 seedlings. Radioactivity was measured 6 h after application of 3 H-IAA in root segments after excision of the apical ≈ 1 mm of the root tip. Values shown are the geometric mean (AE standard deviation, SD) for at least 30 seedlings. The amount of auxin transported into each root segment for Col-0 and eir1-4 was compared by ANOVA at P < 0.05. cpm, counts per minute. B-E Maximum intensity Z-stack projection images of 5 DAG old Col-0 and eir1-4 roots expressing the DII-Venus auxin signaling reporter 12 HAT to ammonium (B and D) or nitrate (C and E) supplemented media. "e" and "c" mark epidermis and cortex, respectively. Scale bar = 50 µm. Graphs denote normalized relative auxin levels at the respective positions. Lines represent polynomial regression fit with 95% confidence band. Data are derived from measurements of n = 8 (ammonium) and n = 10 (nitrate) roots of Col-0 and n = 10 roots of eir1-4 per condition.
Source data are available online for this figure.
The EMBO Journal Krisztina € Otv€ os et al

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The EMBO Journal e106862 | 2020 ª 2020 The Authors A RT-qPCR analysis of PIN2 expression normalized to UBQ10 (AT4G05320) levels in Col-0 roots 1, 6 and 48 HAT to ammonium or nitrate. As a positive control, expression of ANR1 (nitrate responsive MADS-box transcription factor) was quantified. All RT-qPCR reactions were carried out with biological and technical triplicates. Statistical difference was calculated with a t-test (P value ***<0.001). Error bars represent mean AE SD. The experiment was done three times. B PIN2 promoter activity was monitored in pPIN2::nlsGFP and pPIN2::GUS expressing roots 12 HAT to ammonium or nitrate. Scale bars = 50 µm. C Confocal microscopic images of PIN2::PIN2-DENDRA fluorescence in the same area of the root transition zones 12 HAT to ammonium or nitrate before and after photoconversion (0, 20, 120, 180, 240, 300 min). Scale bar = 20 µm. D Multiphoton microscopic image showing polarity changes of PIN2 expression upon nitrate treatment. "e" and "c" denote epidermis and cortex respectively. White arrows mark lateralization of the PIN2-GFP signal in cortex cells (c). Scale bar = 10 µm. ◀ Figure EV5. The role of NRT1.1. in the root growth adaptation to differing nitrogen sources.
A Root growth (RG in µm/min) of roots of Col-0, chl1-5, chl1-9, T101A, and T101D seedlings 12 HAT to ammonium (black) or nitrate (red). Seedlings were transferred at 7 DAG. At least 10 roots/treatment/genotype were measured. The experiment was repeated 3 times. The statistical significance was evaluated with ANOVA at P < 0.01 (**). The box chart components are defined as, box (25-75%), central band (median line), and central box (mean), and the range is within 1.5IQR. B Pseudo-colored, optical longitudinal sections of PIN2 immunostained (yellow signal), 5 DAG roots of chl1-5 and PIN2::PIN2-GFP, eir1-4 expressing roots, 12 HAT to ammonium or nitrate supplemented media. "e" denotes epidermis and "c" cortex, respectively. Scale bar = 30 µm. Box plots display the distribution of the cell membrane-derived anti-PIN2 fluorescence intensity (FI) values (in arbitrary units, a.u.) on ammonium (gray, epidermis (ep) and red, cortex (co), n = 40 membrane) and nitrate (blue, epidermis (ep) and green, cortex (co), n = 40 membrane) grown roots. 4 roots were analyzed/treatment/genotype, and 10 cells per root were quantified. The statistical significance was evaluated with ANOVA at P < 0.05. The box chart components are defined as, box (25-75%), central band (median line), central box (mean), and the range is within 1.5IQR. C Pseudo-colored, longitudinal Z-stacks of PIN1::PIN1-GFP expressing 5 DAG roots, 12 HAT to ammonium or nitrate. Scale bar = 30 µm. Box plots display the distribution of the cell membrane-derived PIN1-GFP fluorescence intensity (FI) values (in arbitrary units, a.u.) on ammonium (black) and red (nitrate). 4 roots were analyzed/ treatment, and 10 cells per root were quantified. The statistical significance was evaluated with ANOVA at P < 0.05. The box chart components are defined as, box (25-75%), central band (median line), and central box (mean), and the range is within 1.5IQR. D Pseudo-colored, longitudinal Z-stacks of PIP2::PIP2-GFP expressing 5 DAG roots, 12 HAT to ammonium or nitrate. Scale bar = 10 µm. Box plots display the distribution of the cell membrane-derived PIP2-GFP fluorescence intensity (FI) values (in arbitrary units, a.u.) on ammonium (black) and red (nitrate). 4 roots were analyzed/ treatment and 10 cells per root were quantified. The statistical significance was evaluated with ANOVA at P < 0.05. The box chart components are defined as, box (25-75%), central band (median line), and central box (mean), and the range is within 1.5IQR.
Source data are available online for this figure.