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Auxin-mediated root branching is determined by the form of available nitrogen

Nature Plants volume 6, pages 1136–1145 (2020)Cite this article

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Abstract

To improve water and nutrient acquisition from the soil, plants can modulate their root system architecture. Despite the importance of changes in root architecture to exploit local nutrient patches occurring in heterogenous soils or after placed fertilization, mechanisms integrating external nutrient signals into the root developmental programme remain poorly understood. Here, we show that local ammonium supply stimulates the accumulation of shoot-derived auxin in the root vasculature and promotes lateral root emergence to build a highly branched root system. Activities of pH and auxin reporters indicate that ammonium uptake mediated by ammonium transporters acidifies the root apoplast, which increases pH-dependent import of protonated auxin into cortical and epidermal cells overlaying lateral root primordia, and subsequently promotes their emergence from the parental root. Thereby, ammonium-induced and H+-ATPase-mediated acidification of the apoplast allows auxin to bypass the auxin importers AUX1 and LAX3. In nitrogen-deficient plants, auxin also accumulates in the root vasculature but a more alkaline apoplast leads to retention of auxin in these tissues and prevents lateral root formation. Our study highlights the impact of externally available nitrogen forms on pH-dependent radial auxin mobility and its regulatory function in organ development.

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Fig. 1: Shoot-derived auxin is critical for higher-order lateral root branching in presence of ammonium.
Fig. 2: N nutrition modulates apoplastic pH and radial auxin distribution in lateral roots.
Fig. 3: Lowering pH restores radial auxin diffusion and higher-order lateral root branching under N deficiency.
Fig. 4: N-dependent response of lateral root development to external auxin.
Fig. 5: Local ammonium promotes lateral root emergence in a LAX3-independent manner.
Fig. 6: Scheme depicting the mechanism underlying ammonium-induced lateral root emergence.

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Data availability

The data that support the findings of this study are available within the article and its supplementary information files or from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank A. T. Fuglsang, University of Copenhagen, and M. J. Bennett, University of Nottingham, for providing mutants and reporter lines. We thank T. Araya, R. F. H. Giehl, D. Heuermann at IPK Gatersleben, as well as C. Oecking, ZMBP Tübingen, for valuable discussions and technical advices. We further thank J. Fuge, A. Bieber, D. Böhmert and C. Bethmann for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft with grant no. WI1728/13-2 to N.v.W. H.T. and K.S.L.P. acknowledge funding support from the National Science Foundation grant no. 1444549 and the USDA NIFA National Needs Training grant no. 2015-38420-23697.

Author information

Author notes

  1. These authors contributed equally: Markus Meier, Ying Liu.

Authors and Affiliations

  1. Molecular Plant Nutrition, Leibniz-Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany

    Markus Meier, Ying Liu & Nicolaus von Wirén

  2. Department of Biochemistry and Molecular Biology, Genetics and Genome Sciences Program, Michigan State University, East Lansing, MI, USA

    Katerina S. Lay-Pruitt & Hideki Takahashi

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  1. Markus Meier
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Contributions

M.M., Y.L., H.T. and N.v.W. designed the experiments. M.M., Y.L. and K.S.L.P. performed the experiments and analysed the data. Y.L., M.M., H.T. and N.v.W. wrote the manuscript.

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Correspondence to Nicolaus von Wirén.

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The authors declare no competing interests.

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Peer review file Nature Plants thanks Tom Beeckman, Chengcai Chu and Yoshiaki Inukai for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Lateral root development of Arabidopsis depends on localized N supply.

a, Experimental setup of vertical and horizontal split-agar plates and definitions of higher-order lateral roots on the HN side. b, Plants grown on vertical split-agar plates with different local N supplies. Ten-day-old Arabidopsis thaliana wild-type (WT, Col-0) plants were transferred to vertical split-agar plates with the first first-order lateral root growing on the high N (HN) side supplemented with 0.8 mM KCl (–N control), 0.8 mM KNO3 (NO3-) or 0.8 mM NH4Cl (NH4+), while the intact primary root remained on the low N (LN) side containing 5 µM KNO3. Images were taken 15 days after transfer. To prevent N leakage from HN to LN, an additional trench was introduced at the bottom. c, d, first-order lateral roots of wild-type (WT) (c) and qko (d) plants grown under different N supplies on the HN side. Lateral roots on the HN side were carefully untangled and scanned 15 days after transfer to local N treatments. Scale bars: 1 cm. e–h, Quantitative assessment of second-order LR length (e), second-order LR density (f), third-order LR length (g) and third-order LR density (h) from roots grown on the HN side. The boxes show the first quartile, median, and third quartile; the whiskers indicate the minimum and maximum values. n = 10 independent biological replicates. Different letters denote significant differences at P < 0.05 according to Tukey’s HSD test.

Source data

Extended Data Fig. 2 Localized ammonium supply promotes lateral root emergence.

a–c, Higher-order lateral root (LR) development of wild-type (Col-0) and qko plants grown under local ammonium supply. Lateral root primordia were classified into three groups according to their developmental stage (I-IV, V-VII and VIII). Ten-day-old plants were transferred to vertical split-agar plates containing 0.8 mM NH4Cl on the HN side. The density of third-order LR primordia and the total LR initiation events on the HN side were determined 7 days after transfer (DAT) (a), 10 DAT (b) and 12 DAT (c). The boxes show the first quartile, median, and third quartile; the whiskers indicate the minimum and maximum values. n = 10 independent biological replicates. Asterisks denote significant differences between WT and qko at * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant, by two-tailed Student’s t-test. d, Relative frequency of LR primordia and emerged LR in wild-type and qko. The relative frequency of LR primordia at different stages is presented as percentage of the total LR initiation events.

Source data

Extended Data Fig. 3 Medium pH affects radial auxin distribution and lateral root branching under local ammonium supply.

a, DII-VENUS fluorescence in response to different medium pH. DII-VENUS reporter lines were grown on vertical split-agar plates containing 0.8 mM NH4Cl on the HN side, where the pH was adjusted to indicated values by 5 mM MES, MOPS or HEPES. Twelve days after transfer, DII-VENUS fluorescence in the branching zone of second-order lateral roots on the HN side was measured by confocal microscopy. Scale bars: 50 µm. b, Quantitative readout of DII-VENUS fluorescence intensity. Bars represent means ± SD, n = 10 independent biological replicates. c, Phenotypes of LR grown on the HN side under different medium pH. Scale bar: 1 cm. d–g, Quantitative assessment of LR traits on the HN side at different pH: second-order LR length (d), second-order LR density (e), third-order LR density (f), and third-order LR density (g). Wild-type seedlings were transferred to vertical split-agar plates with the first first-order lateral root on the HN side exposed to 0.8 mM NH4Cl. The medium pH on the HN side was adjusted to the indicated values by 5 mM MES, MOPS or HEPES, while MES at pH 5.7 was supplied on the LN side of each treatment. Lateral root traits were measured 15 days after transfer. The boxes show the first quartile, median, and third quartile; the whiskers indicate the minimum and maximum values. n = 12 independent biological replicates. Different letters denote significant differences at P < 0.05 according to Tukey’s HSD test.

Source data

Extended Data Fig. 4 Lowering pH recovers higher-order lateral root branching in qko.

a–e, Lateral root development of qko under N deficiency at pH 5.7 or 5.0. Phenotype (a), second-order LR length (b), second-order LR density (c), third-order LR length (d), and third-order LR density (e) of first-order LR of qko. Plants were treated under pH 5.0 or 5.7 adjusted by MES buffer. Lateral root traits in response to pH were determined 7 days after transfer. f–j, Lateral root development of qko under local ammonium at pH 5.7 or 5.0. Phenotype (f), second-order LR length (g), second-order LR density (h), third-order LR length (i), third-order LR density (j) of first-order LR of qko. Ten-day-old qko seedlings were cultured for 7 days under N deficiency (a–e) or 0.8 mM NH4Cl (f–j) at pH 5.7, and then transferred for another 7 days to vertical split-agar plates, in which the first first-order LR on the HN side containing 0.8 mM KCl (a–e) or 0.8 mM NH4Cl (f–j) was exposed to pH 5.0 or 5.7, adjusted by MES buffer. Lateral root traits in response to pH were determined 7 days after transfer. Scale bars: 1 cm. The boxes show the first quartile, median, and third quartile; the whiskers indicate the minimum and maximum values. n = 12 independent biological replicates. Asterisks denote significant differences between pH treatments at ** P < 0.01, *** P < 0.001 by two-tailed Student’s t-test.

Source data

Extended Data Fig. 5 Application of fusicoccin to stimulate H+-ATPase activity promotes lateral auxin diffusion.

a, c, Fluorescence of the DII-VENUS reporter in the branching zone of second-order lateral roots after 4 h (a) or 24 h (c) of fusicoccin treatment. Representative images from 10 plants per treatment are shown. Scale bars: 50 µm. b, d, Quantitative readout of DII-VENUS fluorescence intensity in different cell types after 4 h (b) or 24 h (d) of fusicoccin treatment. Bars represent means ± SD, n = 10 independent biological replicates. Ten-day-old seedlings of DII-VENUS reporter lines were precultured on N-deficient medium for 12 days before transfer to liquid N-deficient medium, in which only the first first-order lateral root was incubated in solution containing either 5 μM fusicoccin or mock medium containing 0.05% (v/v) DMSO.

Source data

Extended Data Fig. 6 Constitutive activation of plasma membrane H+-ATPase promotes higher-order lateral root branching in the fer-4 mutant.

a, Phenotypes of lateral roots exposed to different N treatments on the HN side. Scale bar: 1 cm. b–e, Quantitative assessment of second-order LR length (b), second-order LR density (c), third-order LR density (d), and third-order LR density (e) on the HN side. Wild-type (WT) and fer-4 plants were transferred to vertical split-agar plates with a first-order lateral root growing on the HN side containing either 0.8 mM KCl (-N), 0.8 mM KNO3 or 0.8 mM NH4Cl. Lateral root traits were measured 15 days after transfer. The boxes show the first quartile, median, and third quartile; the whiskers indicate the minimum and maximum values. n = 10 independent biological replicates. Asterisks denote significant differences between WT and fer-4 at * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant, by two-tailed Student’s t-test.

Source data

Extended Data Fig. 7 AHA2 is required for triggering higher-order lateral root branching under local ammonium supply.

a, Phenotypes of lateral roots exposed to 0.8 mM NH4Cl on the HN side. Scale bar: 1 cm. b–e, Quantitative assessment of second-order LR length (b), second-order LR density (c), third-order LR density (d), and third-order LR density (e) on the HN side. Wild-type (WT), aha2–4 and aha2–5 plants were transferred to vertical split-agar plates with a first-order lateral root growing on the HN side containing 0.8 mM NH4Cl. Lateral root traits were measured at 15 days after transfer. The boxes show the first quartile, median, and third quartile; the whiskers indicate the minimum and maximum values. n = 11 independent biological replicates. Different letters represent significant differences at P < 0.05 according to Tukey’s HSD test.

Source data

Extended Data Fig. 8 AUX1 and LAX3 expression in response to local N treatments.

a, Relative transcript levels of AUX1, and LAX3 in lateral roots exposed to different N treatments on the HN side. Col-0 seedlings were transferred to vertical split-agar plates with a first-order lateral root growing on the HN side containing either 0.8 mM KCl (-N), 0.8 mM KNO3 or 0.8 mM NH4Cl. Lateral roots on the HN side were harvested for RNA extraction at 15 days after transfer. Transcript levels were determined by qPCR and normalized by the multiple internal control method using UBQ10 (AT4G05320) and ACTIN2 (AT3G18780) as reference genes. Gene-specific primers are listed in Supplementary Table 1. Bars represent means ± SD, n = 4 independent biological replicates. Different letters represent significant differences among means at P < 0.05, ns = not significant, by Tukey’s HSD test. b, d, Fluorescence of the proAUX1:AUX1:YFP (b) and proLAX3:LAX3:YFP (d) reporters in third-order LR primordia at developmental stage III under local N supplies. Cell walls were stained with propidium iodide. Representative images are from 10 plants per treatment. Scale bars: 50 µm. The seedlings of proAUX1:AUX1:YFP and proLAX3:LAX3:YFP were grown for 12 days on vertical split-agar plates with a first-order lateral root growing on the HN side containing either 0.8 mM KCl (-N), 0.8 mM KNO3 or 0.8 mM NH4Cl. YFP fluorescence of lateral root growing on the HN segment was measured by confocal microscopy. c, e, Quantitative readout of proAUX1:AUX1:YFP fluorescence in vasculature of second-order LR or third-order LR primordia (c) and proLAX3:LAX3:YFP fluorescence in vasculature of second-order LR or the cells of overlaying third-order LR primordia (e). Bars represent means ± SD, n = 10 independent biological replicates.

Source data

Extended Data Fig. 9 Low pH or external supply of NAA recovers lateral root development in the aux1 lax3 mutant.

a–c, Lateral root development of aux1 lax3 mutant plants under N deficiency in dependence of medium pH. Lateral root phenotypes (a), first-order LR length (b), and first-order LR density (c) of aux1 lax3 plants grown under N deficiency. Ten-day-old aux1 lax3 seedlings were transferred to horizontal split-agar plates (containing 0.8 mM KCl in all segments), in which pH in the middle segment was adjusted to 5.0 or 5.7 by MES buffer. Scale bars: 1 cm. Lateral root traits were measured 15 days after transfer. The boxes show the first quartile, median, and third quartile; the whiskers indicate the minimum and maximum values. n = 12 independent biological replicates. Asterisks denote significant differences between pH treatments at *** P < 0.001 according to two-tailed Student’s t-test. d–f, Lateral root development of aux1 lax3 mutant plants under N deficiency with external auxin supplies. Lateral root phenotypes (d), first-order LR length (e), and first-order LR density (f) of aux1 lax3 plants grown under N deficiency. Ten-day-old aux1 lax3 seedlings were transferred to horizontal split-agar plates (containing 0.8 mM KCl in all segments), in which 0.1 µM IAA or NAA was supplied to the middle segment. Scale bars: 1 cm. Lateral root traits were measured 15 days after transfer. The boxes show the first quartile, median, and third quartile; the whiskers indicate the minimum and maximum values. n = 11 independent biological replicates. Different letters represent significant differences at P < 0.05 by Tukey’s HSD test.

Source data

Extended Data Fig. 10 The expression of the cell wall-remodelling gene polygalacturonase (PG) responds to local N treatments.

Seedlings of the reporter line pPG:GUS were transferred to vertical split-agar plates with a first-order lateral root growing on the HN side containing either 0.8 mM KCl (-N), 0.8 mM KNO3 or 0.8 mM NH4Cl. Lateral roots from the HN side were used for GUS staining 12 days after transfer. GUS signals around third-order lateral root primordia at different developmental stages were analysed by DIC microscopy. Asterisks indicate the tip of third-order lateral root primordia. Representative images from 10 plants per treatment are shown. Scale bars: 50 µm.

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Meier, M., Liu, Y., Lay-Pruitt, K.S. et al. Auxin-mediated root branching is determined by the form of available nitrogen. Nat. Plants 6, 1136–1145 (2020). https://doi.org/10.1038/s41477-020-00756-2

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