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A secreted peptide acts on BIN2-mediated phosphorylation of ARFs to potentiate auxin response during lateral root development

Nature Cell Biology volume 16, pages 66–76 (2014)Cite this article

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Abstract

The phytohormone auxin is a key developmental signal in plants. So far, only auxin perception has been described to trigger the release of transcription factors termed AUXIN RESPONSE FACTORs (ARFs) from their AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) repressor proteins. Here, we show that phosphorylation of ARF7 and ARF19 by BRASSINOSTEROID-INSENSITIVE2 (BIN2) can also potentiate auxin signalling output during lateral root organogenesis. BIN2-mediated phosphorylation of ARF7 and ARF19 suppresses their interaction with AUX/IAAs, and subsequently enhances the transcriptional activity to their target genes LATERAL ORGAN BOUNDARIES-DOMAIN16 (LBD16) and LBD29. In this context, BIN2 is under the control of the TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)–TDIF RECEPTOR (TDR) module. TDIF-initiated TDR signalling directly acts on BIN2-mediated ARF phosphorylation, leading to the regulation of auxin signalling during lateral root development. In summary, this study delineates a TDIF–TDR–BIN2 signalling cascade that controls regulation of ARF and AUX/IAA interaction independent of auxin perception during lateral root development.

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Figure 1: BIN2 positively regulates LR development.
Figure 2: BR plays a minor role in BIN2-mediated LR development.
Figure 3: BIN2-mediated signalling is integrated into ARF7/19 during LR development.
Figure 4: BIN2 directly phosphorylates ARF7.
Figure 5: BIN2 attenuates the interaction of ARF7 with AUX/IAAs and enhances its transcriptional activity, leading to activation of auxin response.
Figure 6: BIN2 increases DNA-binding capacity of ARF7 and ARF19.
Figure 7: TDIF–TDR module is directly linked to BIN2-mediated auxin signal activation in LR development.

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Acknowledgements

The authors thank D. Weijers for critical reading of the manuscript and useful suggestions. This work was supported by grants from the Advanced Biomass R&D Center (2010-0029720) and the National Research Foundation (20110000212) funded by the Korean Ministry of Education, Science and Technology. H.R. was supported by grants from the Next-Generation BioGreen 21 Program (PJ009516). H.C. and S.H. were recipients of a Brain Korea 21 fellowship. I.D.S. was supported by a BBSRC David Phillips Fellowship (BB_BB/H022457/1) and a Marie Curie European Reintegration Grant (PERG06-GA-2009-256354).

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  1. Hyunwoo Cho and Hojin Ryu: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea

    Hyunwoo Cho, Hojin Ryu, Joonghyuk Park, Soeun Han & Ildoo Hwang

  2. School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Korea

    Sangchul Rho & Daehee Hwang

  3. Division of Plant and Crop Sciences, School of Biosciences University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK

    Kristine Hill, Stephanie Smith, Malcolm J. Bennett & Ive De Smet

  4. Centre for Plant Integrative Biology, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK

    Kristine Hill & Malcolm J. Bennett

  5. Integrative Plant Biology Division, Department of Plant Systems Biology, VIB, Technologiepark 927, Ghent 9052, Belgium

    Dominique Audenaert, Tom Beeckman & Ive De Smet

  6. Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, Ghent 9052, Belgium

    Dominique Audenaert, Tom Beeckman & Ive De Smet

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Contributions

H.C., H.R., S.H., J.P., K.H. and S.S. performed experiments. H.C., H.R., I.D.S. and I.H. designed experiments and analysed the data. D.A. analysed gene expression. S.R. and D.H. performed LC–MS/MS analysis. H.C., H.R., T.B., M.J.B., I.D.S. and I.H. wrote the manuscript.

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Correspondence to Ildoo Hwang.

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Integrated supplementary information

Supplementary Figure 1 The expression pattern of BIN2, ARF7 and ARF19 in root.

(a) BIN2 expression in basal meristem and LRP at different stages of lateral root development. Note that the expression is restricted to the basal part of LRP at stages IV-VII. Scale bar, 50 μm. (b,c) ARF7 (b) and ARF19 (c) expression in LRP at early stages of lateral root development. GUS activity driven by the ARF7 or ARF19 promoter was observed in early stages of LRP. Scale bar, 50 μm.

Supplementary Figure 2 BIN2 enhances auxin-response in plants.

(a) Three-day-old seedlings of pDR5-GUS and pDR5-GUS / bin2-1-gof grown on 10 μM NPA-containing medium were transferred to 1 μM NAA-containing medium, and further treated for 1, 3, and 5 h. The GUS activity of the seedlings was measured. Error bars indicate SEM (n = 3, 20 plants per n = 1, * for P<0.05 by student’s t-test). (b) The expression levels of LBD16 and LBD29 in 10-day-old bri1-116 and bin2-1-gof (+/+) mutants were determined by quantitative RT-PCR (n = 4, 10 plants per n = 1, * for P<0.05 by student’s t-tests). (c,d) The expression levels of LBD16 (left panel) and LBD29 (right panel) in 10-day-old WT and bin2-1-gof (c), dwf12-1D-gof (d), and bin2-3 bil1 bil2 mutants were determined by quantitative RT-PCR after auxin treatment for the indicated times. Error bars indicate SEM (n = 3, 10 plants per n = 1, * for P<0.05 by student’s t-tests). (e) Bikinin treatment represses LBD16 and LBD29 expression. Error bars indicate SEM (n = 3, 10 plants per n = 1, * for P<0.05 by student’s t-tests). (f) BIN2 enhances the activity of pGH3-LUC auxin-responsive reporter in a dose-dependent manner. Protoplasts were co-transfected with pGH3-LUC, indicated amount of BIN2-HA, and pUBQ10-GUS (internal control). Error bars indicate the SEM (n = 4, 20,000 cells per n = 1, * for P<0.05, ** for P<0.01 by student’s t-test). Statistics source data found in Supplementary Table 2. Details on the statistical analysis are found in Methods.

Supplementary Figure 3 BIN2 and BIL1 are involved in lateral root formation.

(a) BIN2 and BIL1 enhance auxin-responsive pGH3-LUC activity. Protoplasts were cotransfected with pUBQ10-GUS (internal control), pGH3-LUC and an effector plasmid harboring BIN2, BIL1 or BIL2. Error bars indicate SEM (n = 4, 20,000 cells per n = 1, * for P<0.05 by student’s t-test). (b) Isolation of bil1 knockout mutant. The transcripts of BIL1 in 7-day-old wild type (Ws-2) and bil1 (FLAG_426B01) were determined by semi-quantitative PCR. (c) Lateral root densities of twelve-day-old seedlings of WT (Ws-2), bin2-3-lof, bil1, bil1 bin2-3 and bin2-3 bil1 bil2 mutant. E, emerged lateral roots; NE, non-emerged LRP. Error bars indicate SEM (n = 15 plants, * for P<0.05, ** for P<0.01 by student’s t-test). (d) The transcripts of ARF7 in WT (Col-0), arf7-1 and arf7-1 bin2-1-gof were determined by semi-quantitative PCR. (e) The transcripts of ARF7 and ARF19 in WT (Col-0) and arf7-1 arf19-1 bin2-1-gof were determined by semi-quantitative PCR. Tubulin and Actin2 genes served as input controls. Details on the statistical analysis are found in Methods.

Supplementary Figure 4 BIN2 does not interact with AUX/IAA proteins.

HA-tagged BIN2 was expressed in protoplasts. Protoplast lysates were incubated with GST-IAA1, GST-IAA3, or GST-IAA19 proteins for 1 h, and then precipitated with glutathione sepharose 4B. BIN2 proteins were detected with anti-HA antibody (upper and middle panels), and GST-tagged IAA1, IAA3, or IAA19 proteins were visualized by Coomassie blue staining (lower panel).

Supplementary Figure 5 BIN2-mediated phosphorylation of ARF7 attenuates its interaction with IAA1, IAA3, IAA14 and IAA18.

Protoplast lysates overexpressing ARF7-HA or ARF7-HA and BIN2-HA were incubated with purified GST-tagged IAA1, IAA3, IAA14, or IAA18 proteins and pulled down with glutathione sepharose 4B. The pulled-down proteins were analyzed with anti-HA antibody. The GST protein was used as a negative control. An asterisk indicates GST-IAAs proteins.

Supplementary Figure 6 (a) BIN2-mediated phosphorylation of ARF7 results in increased binding of IAA19 with TIR1.

Two more independent pull down experiments conducted in Fig. 6a are presented. HA-tagged ARF7 and TIR1 were co-transfected into protoplasts with or without BIN2-HA. After 6 h of incubation, protoplast lysates were incubated with purified GST-IAA19 proteins and 10 μM of MG132 for 1 h, and then pulled down with glutathione sepharose 4B. GST-IAA19-bound proteins were visualized with anti-HA antibody. GST protein was included as a negative control. Asterisks indicate GST-IAA19 proteins. The levels of GST-IAA19 pulled-down proteins were normalized to those of the input proteins. The normalized intensities of the pulled-down proteins in the absence of BIN2 were set to 1, and the relative protein intensities were presented under the corresponding protein bands. (b) Relative DII-VENUS intensities in seedlings of mock or 15 μMbikinin treated WT (Col-0). (n = 20 plants).

Supplementary Figure 7 Brassinosteroid is not involved in BIN2-mediated ARF7 activation.

(a,b) Brassinosteroid does not affect the phosphorylation status of ARF7. Seven-day-old transgenic seedlings harboring 35S-ARF7-HA (left panel), 35S-ARF7-HA / dwf12-1D-gof (b), or 35S-BES1-HA (a positive BR-responsive control, right panel) were incubated with or without 20 nM of epi-BL for 6 and 12 h. The phosphorylation status of HA-tagged ARF7 and BES1 was determined as the protein mobility shift with anti-HA antibody. (c) Bikinin inhibits BIN2-induced phosphorylation of ARF7. ARF7-HA was co-transfected with BIN2-HA into protoplasts and incubated with or without 30 μM of bikinin for 6 h. ARF7 proteins were visualized with anti-HA antibody. (d) Brassinosteroid could not suppress the BIN2 actions for enhancing the transcriptional activity of ARF7. ARF7-HA, pGH3-LUC and pUBQ10-GUS were cotransfected into protoplasts with or without BIN2-HA. The protoplasts were incubated with or without 1 μM epi-BL and 30 μM bikinin for 6 h. Error bars indicate SEM (n = 4, 20,000 cells per n = 1).

Supplementary Figure 8 Full scan data of immunoblot and in vitro kinase assay.

Supplementary Table 1 Primer combinations for quantitative PCR.
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Cho, H., Ryu, H., Rho, S. et al. A secreted peptide acts on BIN2-mediated phosphorylation of ARFs to potentiate auxin response during lateral root development. Nat Cell Biol 16, 66–76 (2014). https://doi.org/10.1038/ncb2893

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