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Design principles of a minimal auxin response system

Nature Plants volume 6, pages 473–482 (2020)Cite this article

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Abstract

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1,2,3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development1,3,4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems.

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Fig. 1: Functional diversity in DNA binding among MpARFs.
Fig. 2: Activation and direct repression by MpARFs.
Fig. 3: Dual function of the PB1 domain.
Fig. 4: A/B-ARF antagonism defines a minimal auxin response model.

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Data availability

All materials generated in this study are freely available upon request from the corresponding author. All data are available in the main text or the supplementary materials. The crystallographic data are available from the RCSB PDB (accession number 6SDG), and the RNA-seq data are available from the NCBI SRA under the project accession number PRJNA554398 (http://www.ncbi.nlm.nih.gov/bioproject/554398).

Code availability

The Python script used to calculate the residues in dimer interface is available at http://www.protein.osaka-u.ac.jp/rcsfp/supracryst/suzuki/jpxtal/Katsutani/InterfaceResidues.py.

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Acknowledgements

We thank the XALOC staff at the synchrotron ALBA and V. P. Carrillo, Carrasco, S. Kiryu, M. Katayama and L. Olijslager for experimental support, D. Gadella and K. Hiratsuka for providing materials, and O. Leyser for comments in the manuscript. This work was supported by an EMBO Long-term Fellowship (ALTF 415-2016) to H.K., a PhD fellowship from the Graduate School Experimental Plant Sciences to J.H. and D.W., a VICI grant (no. 865.14.001) from the Netherlands Organization for Scientific Research (NWO) to D.W., grants from the Ministry of Economy and Competitiveness of the Spanish Government (nos BIO2016-77883-C2-2-P and FIS2015-72574-EXP) (AEI/FEDER,EU) to D.R.B., an ALW-open grant (no. ALWOP.402) from the NWO to J.W.B., JSPS/MEXT KAKENHI (grant nos 19K23751 to H.K., 18J12698 to H.S, 19K016166 to Y.Y., 17H06472 to K.I. 18H04836 to R.N. and 25113009, 15K21758 and 19H05675 to T.K.) and SPIRITS 2017 of Kyoto University to R.N.

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Author notes

  1. These authors contributed equally: Sumanth K. Mutte, Hidemasa Suzuki, Isidro Crespo, Shubhajit Das, Tatyana Radoeva, Mattia Fontana.

Authors and Affiliations

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

    Hirotaka Kato, Sumanth K. Mutte, Shubhajit Das, Tatyana Radoeva, Mattia Fontana, Willy van den Berg, Simon Lindhoud, Jan Willem Borst & Dolf Weijers

  2. Graduate School of Science, Kobe University, Kobe, Japan

    Hirotaka Kato & Kimitsune Ishizaki

  3. Graduate School of Biostudies, Kyoto University, Kyoto, Japan

    Hidemasa Suzuki, Yoshihiro Yoshitake, Emi Hainiwa, Ryuichi Nishihama & Takayuki Kohchi

  4. Alba Synchrotron, Cerdanyola del Vallès, Barcelona, Spain

    Isidro Crespo & D. Roeland Boer

  5. Laboratory of Biophysics, Wageningen University, Wageningen, The Netherlands

    Mattia Fontana & Johannes Hohlbein

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Contributions

H.K., R.N., T.K. and D.W. conceptualized the project. H.K., S.K.M., H.S., I.C., T.R., S.D., M.F., E.H., W.v.d.B. and S.L. conducted the investigation. H.K., S.K.M., I.C. and M.F. conducted the formal analysis. J.H., D.R.B., R.N., T.K. and D.W. supervised the project. K.I., J.H., R.N., T.K., J.W.B. and D.W. acquired the funding. H.K. and D.W. wrote the original draft of the paper. All authors reviewed and edited the paper.

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Correspondence to Dolf Weijers.

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Extended data

Extended Data Fig. 1 Transgene expression and phenotypes of DBD swap lines.

a, Expression of transgenic transcripts in gemmalings of three independent proARF1-ARF1, proARF1-ARF2 and proARF1-ARF3 lines, measured by qRT-PCR using the common 5’-UTR fragment. Circles indicate each data point for three technical replicates. ND: not detected in Tak-1 wild-type and arf1-4 mutant. b, Ten-day-old gemmalings and c, immature gemmae of Tak-1 wild-type, Mparf1-4, proMpARF1:ARF111 (111), proMpARF1:ARF211 (211) and proMpARF1:ARF311 (311). Bars = 2 mm in (b) and 0.1 mm in (c). The experiment was repeated twice with similar results.

Extended Data Fig. 2 Gemmae phenotypes in Mparf1 and Mparf3 mutants.

Optical medial sections through early- (top row) and late-stage (bottom row) gemmae from Tak-1, Mparf1-4 and Mparf3ge1-1 plants. Medial sections were taken from full 3D stacks of gemmae in which cell walls were labeled using mPS-PI staining. The positions of the future apical notches are indicated by red arrowheads in Tak-1. Note that these indentations are missing in both mutants. Bars are 20 µm in all panels. The experiment was repeated twice with similar results.

Extended Data Fig. 3 Gene Ontology analysis of genes misexpressed in arf1 and arf3 gemmae.

GO categories ‘Biological process’ and ‘Molecular function’ were shown for downregulated genes as directed graphs with least- and most- enriched categories are shown in yellow and red blocks, respectively, as well as in the table underneath the figures. Note the different categories enriched in arf1 and arf3. P-values (table) are derived from a Fisher’s exact test.

Extended Data Fig. 4 Phenotypes induced by Middle Region domain swaps in Tak-1 background.

Ten-day-old gemmalings of Tak-1 wild-type, and 2 independent lines each for proMpARF1:ARF121 (121) and proMpARF1:ARF131 (131) Bar = 1 mm. The experiment was repeated four times with similar results.

Extended Data Fig. 5 Expression of MpTPL and MpARFs for FRET-FLIM.

Fluorescence of MpTPL-mNeongreen (FRET donor; top row), either co-expressed with fusions of mScarlet-I (FRET acceptor; middle row), to empty vector or with MpARF1-mScarlet-I, MpARF2-mScarlet-I or MpARF2(LFG-AAA)-mScarlet-I. Bottom panel shows overlay of mNeongreen, mScarlet-I and chloroplast autofluorescence signals. Note that all fusion proteins localize to nuclei. Bar is 5 µm. The experiment was repeated twice with similar results and with 15, 10, 10 and 15 protoplasts (left to right).

Extended Data Fig. 6 Biological significance of the MpARF LFG motifs.

a, Ten-day-old gemmalings of proMpARF1:ARF11Δ1 (11Δ1), proMpARF1:ARF12Δ1 (12Δ1) and proMpARF1:ARF13Δ1 (13Δ1) lines. The experiment was repeated twice with similar results. b, Relative qPCR expression of WIP gene in Tak-1 wild-type, proMpARF1:ARF12Δ1 (12Δ1) and proMpARF1:ARF13Δ1 (13Δ1) gemmalings, 0, 1, 2 and 4 hours after treatment with 10 µM 2,4-D. The experiment was performed once with three technical replicates. c, Relative qPCR expression of YUC2 gene in Tak-1 wild-type, Mparf1-4 and proMpARF1:ARF11Δ1 (11Δ1) gemmalings, after a 1-hour treatment with control media or with 10 µM 2,4-D. n = 3 technical replicates. The experiment was repeated twice with similar results. Bar in (a) = 2 mm.

Extended Data Fig. 7 Docking of MpARF and MpIAA PB1 domains.

Best fit structural models of molecular docking of PB1 domains of MpARF1 (top), MpARF2 (middle) and MpARF3 (bottom) with MpIAA. Residues of positive (K, Lysine) and negative (OPCA) faces are indicated as sticks.

Extended Data Fig. 8 Antagonistic action of MpARF1 and MpARF2.

Fourteen-day-old gemmalings of Tak-1, proEF:MpARF1, proEF:MpARF2, and proEF:MpARF1 proEF:MpARF2 transgenic lines grown on control medium, or on media containing 1, 3 or 10 µM 2,4-D. The proEF:MpARF1 proEF:MpARF2 line was generated by introducing the proEF:MpARF1 cassette into the presented proEF:MpARF2 line. Bar = 1 cm. The experiment was repeated twice with similar results.

Extended Data Fig. 9 Conditional MpARF2 inactivation.

a, Design of transgene for inducible removal of MpARF2. A CRISPR-resistant MpARF2 version (ARF2m; through engineered silent mutations) is flanked by loxP sites and an upstream EF promoter and a downstream tdTomato-NLS gene, in a construct that also harbors a DEX-inducible Cre-GR version driven from a heat-inducible MpHSP17.8A1 promoter (proHSP). Following heat shock, and in the presence of DEX, Cre-GR will recombine the two loxP sites and excise the ARF2m locus. Cells in which recombination occurred are marked by proEF:tdTomato-NLS expression. b, CRISPR/Cas9-induced mutations in the endogenous MpARF2 gene in Mparf2cko genetic background. Two different single-base insertions were recovered, generating two independent Mparf2cko alleles. c, Phenotypes of 8-day-old gemmalings of Tak-1, Mparf2-1cko or Mparf2-2cko lines grown on control medium or with 1 µM DEX after a heat shock on day 1. The experiment was repeated four times with similar results. d, PCR amplicons detecting the endogenous ARF2 (upper band) and ARF2m (lower band) genes in Tak-1 and in 3 independent transgenic lines harboring ARF2m (#11, #14, #18; prior to introduction of the CRISPR/Cas9 construct), either grown on control media, or on 1 µM DEX after a heat shock. The experiment was repeated twice with similar results. e, tdTomato expression (lower panels) and appearance (top panels) of 3-day-old Mparf2cko gemmalings grown on control medium or with 1 µM DEX after a heat shock on day 1. The experiment was repeated four times with similar results. Bars are 1 mm in (c) and 0.2 mm in (e).

Extended Data Fig. 10 Single-molecule FRET analysis of ARF-DNA interaction.

a, Schematic representation of the ER7 DNA oligonucleotide used in single-molecule FRET experiments, harboring two inverted ARF binding sites (indicated in bold lettering and with a red arrow on top- and bottom-strand, respectively). The bottom-strand was 5’ biotinylated (indicated with an orange hexagon, labeled B) to facilitate immobilization on cover slips. FRET-compatible Cy3B (green circle) and ATTO647N (magenta circle) labels were attached immediately upstream of each ARF binding site indicated with yellow). b, Simulations of accessible volumes of the Cy3B and ATTO647N dyes on the ER7 oligonucleotide in the absence (left) or presence (right) of MpARF2 DBD (based on the AtARF2-ER7 complex, PDB ID: 6SDG). Note that dye clouds are constrained upon protein binding, predicting reduced FRET efficiency. c, Histograms showing FRET efficiency (x-axis) and Cy3B/ATTO6437N stoichiometry (y-axis) of the immobilized ER7 oligonucleotide incubated without MpARF1 DBD (left) or with 256 nM MpARF1 DBD (right). Each dot represents a single DNA molecule. Note the shift in FRET efficiency induced by protein binding. d,e, Distribution histograms of FRET efficiency (x-axis) of the labeled and immobilized ER7 oligonucleotide in the presence of increasing concentrations (0-256 nM) of MpARF1 DBD (d) or MpARF2 DBD (e). Each titration series is followed by an incubation without protein to validate recovery to unbound state. Histograms show fits of the two FRET states representing unbound and bound states, and indicate percentages of DNA molecules in each state.

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Kato, H., Mutte, S.K., Suzuki, H. et al. Design principles of a minimal auxin response system. Nat. Plants 6, 473–482 (2020). https://doi.org/10.1038/s41477-020-0662-y

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