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Reporters for sensitive and quantitative measurement of auxin response

Nature Methods volume 12, pages 207–210 (2015)Cite this article

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A Corrigendum to this article was published on 29 October 2015

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Abstract

The visualization of hormonal signaling input and output is key to understanding how multicellular development is regulated. The plant signaling molecule auxin triggers many growth and developmental responses, but current tools lack the sensitivity or precision to visualize these. We developed a set of fluorescent reporters that allow sensitive and semiquantitative readout of auxin responses at cellular resolution in Arabidopsis thaliana. These generic tools are suitable for any transformable plant species.

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Figure 1: DR5v2 sensitively reports auxin response.
Figure 2: R2D2, a semiquantitative and rapid auxin-input reporter.

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Change history

  • 24 September 2014

    In the version of this article initially published, it was stated that all transgenic lines were created in the Arabidopsis Col-0 ecotype. However, they were actually generated in the Columbia-Utrecht (Col-utr) ecotype. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank T. Laux (Universität Freiburg) for plasmids and B. de Rybel for helpful comments on the manuscript. This work was supported by grants from the European Research Council (ERC; CELLPATTERN; contract number 281573) and the Netherlands Organization for Scientific Research (NWO; ALW-820.02.019) to D.W. and Human Frontier Science Program (HFSP; research grant RGP0054-2013) and Agence Nationale de la Recherche (ANR; AuxiFlo; grant ANR-12-BSV6-0005) to T.V.

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Author notes

  1. Saiko Yoshida

    Present address: Present address: Institute of Science and Technology, Klosterneuburg, Austria.,

Authors and Affiliations

  1. Laboratory of Biochemistry, Wageningen University, Wageningen, the Netherlands

    Che-Yang Liao, Wouter Smet, Saiko Yoshida & Dolf Weijers

  2. Laboratoire de Reproduction et Développement des Plantes, CNRS, Institut National de la Recherche Agronomique, Ecole Normale Superieure Lyon, Lyon, France

    Geraldine Brunoud & Teva Vernoux

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  1. Che-Yang Liao
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Contributions

C.-Y.L. generated all transgenic lines with the exception of RPS5A-DII-Venus lines, which were generated by G.B. All imaging was performed by C.-Y.L. and W.S. S.Y. contributed to analysis of DII-Venus lines. D.W. and T.V. supervised the project. C.-Y.L. and D.W. conceived of the study and wrote the paper with input from all authors.

Corresponding author

Correspondence to Dolf Weijers.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Overview comparison of DR5 and DR5v2 activity in root tip.

Maximal projection of propidium iodine stained root of (a) DR5:: n3eGFP and (b) DR5v2:: n3eGFP reporter lines. Scale bars are 10 µm.

Source data

Supplementary Figure 2 Performance of primers against tandem repeated reporter genes.

qRT-PCR of serial diluted serial diluted pGIIM/DR5v2::ntdTomato-DR5::n3eGFP plasmid with primers used in this study. Bars indicate standard error from the mean (n=3).

Supplementary Figure 3 Response of DR5 and DR5v2 to external auxin.

Fluorescent signal intensity of n3xGFP (top row) and ntdTomato (bottom row) in DR5v2::ntdTomato-DR5::n3eGFP root tips following a 12-hour co-treatment of 10 µM NPA and the indicated concentrations of IAA. Detector gain was saturated for each channel separately at the highest signal intensity of the 1000 nM IAA treated root, and all other images were acquired using these same settings. Signal intensity is displayed as a false color scale. Scale bars are 10 µm.

Source data

Supplementary Figure 4 pRPS5a::DII:Venus and pRPS5a::mDII:Venus root tips.

(a) pRPS5a:: DII: Venus and (b) pRPS5a:: mDII: Venus. Scale bars are 10 µm.

Source data

Supplementary Figure 5 DR5v2-n3GFP and R2D2 heart-stage embryos.

Scale bars are 10 µm.

Supplementary Figure 6 Quantification of R2D2 gradients.

Normalized ntdTomato/n3xVenus signal ratio in nuclei at increasing distance from the QC (see dashed lines in image on the right). Cell 1 corresponds to the first daughter of the initial for each cell file. Red/yellow ratio was set to “1” in cell 1 for each cell file. Bars indicate standard error from the mean (n>30 cell files per tissue). Scale bars are 10 µm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note 1 (PDF 1731 kb)

R2D2 activity upon auxin treatment

Overlay of red and green fluorescence in a root after treatment with 1 μM IAA during 33 minutes (1 frame every 3 minutes). (AVI 8450 kb)

R2D2 activity upon control treatment

Overlay of red and green fluorescence in a root after treatment with control medium during 33 minutes (1 frame every 3 minutes). (AVI 8450 kb)

Source data

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Liao, CY., Smet, W., Brunoud, G. et al. Reporters for sensitive and quantitative measurement of auxin response. Nat Methods 12, 207–210 (2015). https://doi.org/10.1038/nmeth.3279

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