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DWARF14 is a non-canonical hormone receptor for strigolactone

Abstract

Classical hormone receptors reversibly and non-covalently bind active hormone molecules, which are generated by biosynthetic enzymes, to trigger signal transduction. The α/β hydrolase DWARF14 (D14), which hydrolyses the plant branching hormone strigolactone and interacts with the F-box protein D3/MAX2, is probably involved in strigolactone detection1,2,3. However, the active form of strigolactone has yet to be identified and it is unclear which protein directly binds the active form of strigolactone, and in which manner, to act as the genuine strigolactone receptor. Here we report the crystal structure of the strigolactone-induced AtD14–D3–ASK1 complex, reveal that Arabidopsis thaliana (At)D14 undergoes an open-to-closed state transition to trigger strigolactone signalling, and demonstrate that strigolactone is hydrolysed into a covalently linked intermediate molecule (CLIM) to initiate a conformational change of AtD14 to facilitate interaction with D3. Notably, analyses of a highly branched Arabidopsis mutant d14-5 show that the AtD14(G158E) mutant maintains enzyme activity to hydrolyse strigolactone, but fails to efficiently interact with D3/MAX2 and loses the ability to act as a receptor that triggers strigolactone signalling in planta. These findings uncover a mechanism underlying the allosteric activation of AtD14 by strigolactone hydrolysis into CLIM, and define AtD14 as a non-canonical hormone receptor with dual functions to generate and sense the active form of strigolactone.

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Figure 1: Overall structure of AtD14–D3–ASK1 in complex with an active SL intermediate.
Figure 2: Allosteric activation of AtD14 for the open-to-closed conformational transition.
Figure 3: Structural analyses of the AtD14-D3 interaction.
Figure 4: AtD14(G158E) hydrolyses SL but fails to efficiently bind MAX2 for triggering SL signalling.

Accession codes

Data deposits

Crystallographic coordinates have been deposited in the Protein Data Bank under accession numbers 5HZG (CLIM–AtD14–D3–ASK1) and 5HYW (D3–ASK1).

References

  1. Hamiaux, C. et al. DAD2 is an α/β hydrolase likely to be involved in the perception of the plant branching hormone, strigolactone. Curr. Biol. 22, 2032–2036 (2012)

    CAS  Article  Google Scholar 

  2. Nakamura, H. et al. Molecular mechanism of strigolactone perception by DWARF14. Nat. Commun. 4, 2613 (2013)

    ADS  Article  Google Scholar 

  3. Zhao, L.-H. et al. Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3. Cell Res. 25, 1219–1236 (2015)

    CAS  Article  Google Scholar 

  4. Norman, A. W. & Henry, H. L. Hormones. (Elsevier Science, 2014)

  5. Tan, X. et al. Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446, 640–645 (2007)

    CAS  ADS  Article  Google Scholar 

  6. Murase, K., Hirano, Y., Sun, T. P. & Hakoshima, T. Gibberellin-induced DELLA recognition by the gibberellin receptor GID1. Nature 456, 459–463 (2008)

    CAS  ADS  Article  Google Scholar 

  7. Santiago, J. et al. The abscisic acid receptor PYR1 in complex with abscisic acid. Nature 462, 665–668 (2009)

    CAS  ADS  Article  Google Scholar 

  8. Sheard, L. B. et al. Jasmonate perception by inositol-phosphate-potentiated COI1–JAZ co-receptor. Nature 468, 400–405 (2010)

    CAS  ADS  Article  Google Scholar 

  9. Hothorn, M. et al. Structural basis of steroid hormone perception by the receptor kinase BRI1. Nature 474, 467–471 (2011)

    CAS  Article  Google Scholar 

  10. Hothorn, M., Dabi, T. & Chory, J. Structural basis for cytokinin recognition by Arabidopsis thaliana histidine kinase 4. Nat. Chem. Biol. 7, 766–768 (2011)

    CAS  Article  Google Scholar 

  11. Gomez-Roldan, V. et al. Strigolactone inhibition of shoot branching. Nature 455, 189–194 (2008)

    CAS  ADS  Article  Google Scholar 

  12. Umehara, M. et al. Inhibition of shoot branching by new terpenoid plant hormones. Nature 455, 195–200 (2008)

    CAS  ADS  Article  Google Scholar 

  13. Kretzschmar, T. et al. A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching. Nature 483, 341–344 (2012)

    CAS  ADS  Article  Google Scholar 

  14. Akiyama, K., Matsuzaki, K. & Hayashi, H. Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature 435, 824–827 (2005)

    CAS  ADS  Article  Google Scholar 

  15. Conn, C. E. et al. Plant evolution. Convergent evolution of strigolactone perception enabled host detection in parasitic plants. Science 349, 540–543 (2015)

    CAS  ADS  Article  Google Scholar 

  16. Toh, S. et al. Structure-function analysis identifies highly sensitive strigolactone receptors in Striga. Science 350, 203–207 (2015)

    CAS  ADS  Article  Google Scholar 

  17. Tsuchiya, Y. et al. Parasitic plants. Probing strigolactone receptors in Striga hermonthica with fluorescence. Science 349, 864–868 (2015)

    CAS  ADS  Article  Google Scholar 

  18. Stirnberg, P., van De Sande, K. & Leyser, H. M. MAX1 and MAX2 control shoot lateral branching in Arabidopsis. Development 129, 1131–1141 (2002)

    CAS  PubMed  Google Scholar 

  19. Waters, M. T. et al. Specialisation within the DWARF14 protein family confers distinct responses to karrikins and strigolactones in Arabidopsis. Development 139, 1285–1295 (2012)

    CAS  Article  Google Scholar 

  20. Chevalier, F. et al. Strigolactone promotes degradation of DWARF14, an α/β hydrolase essential for strigolactone signaling in Arabidopsis. Plant Cell 26, 1134–1150 (2014)

    CAS  Article  Google Scholar 

  21. Arite, T. et al. d14, a strigolactone-insensitive mutant of rice, shows an accelerated outgrowth of tillers. Plant Cell Physiol. 50, 1416–1424 (2009)

    CAS  Article  Google Scholar 

  22. Ishikawa, S. et al. Suppression of tiller bud activity in tillering dwarf mutants of rice. Plant Cell Physiol. 46, 79–86 (2005)

    CAS  Article  Google Scholar 

  23. Johnson, X. et al. Branching genes are conserved across species. Genes controlling a novel signal in pea are coregulated by other long-distance signals. Plant Physiol. 142, 1014–1026 (2006)

    CAS  Article  Google Scholar 

  24. Zhao, L. H. et al. Crystal structures of two phytohormone signal-transducing α/β hydrolases: karrikin-signaling KAI2 and strigolactone-signaling DWARF14. Cell Res. 23, 436–439 (2013)

    CAS  Article  Google Scholar 

  25. Jiang, L. et al. DWARF 53 acts as a repressor of strigolactone signalling in rice. Nature 504, 401–405 (2013)

    CAS  ADS  Article  Google Scholar 

  26. Zhou, F. et al. D14–SCFD3-dependent degradation of D53 regulates strigolactone signalling. Nature 504, 406–410 (2013)

    CAS  ADS  Article  Google Scholar 

  27. Wang, L. et al. Strigolactone signaling in Arabidopsis regulates shoot development by targeting D53-Like SMXL repressor proteins for ubiquitination and degradation. Plant Cell 27, 3128–3142 (2015)

    CAS  Article  Google Scholar 

  28. Nelson, D. C. et al. F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 108, 8897–8902 (2011)

    CAS  ADS  Article  Google Scholar 

  29. Umehara, M. et al. Structural requirements of strigolactones for shoot branching inhibition in rice and Arabidopsis. Plant Cell Physiol. 56, 1059–1072 (2015)

    CAS  Article  Google Scholar 

  30. Kagiyama, M. et al. Structures of D14 and D14L in the strigolactone and karrikin signaling pathways. Genes Cells 18, 147–160 (2013)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank the staff (J. He, Q. Wang and F. Yu) at the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U and the staff at Tsukuba Photon Factory beamline NE3A for help with data collection. We also thank G. Liu (School of Pharmaceutical Sciences), Z. Yin and H. Fu (Department of Chemistry) at Tsinghua University, B. Xia and P. Ding (School of Life Sciences), T. Luo (College of Chemistry and Molecular Engineering) at Peking University for valuable suggestions on chemistry analysis; Y. Ding, W. Wang and Y. Tian (Drug Facility, School of Pharmaceutical Sciences) at Tsinghua University for help with D-OH quantitation. This work was supported by the National Natural Science Foundation of China (Grant no. 91417302, 81322023, 31421001 and 91335204), the Ministry of Science and Technology (grant number 2013CB911100 and 2016YFA0500500), and the Ministry of Agriculture (grant number 2014ZX08011006).

Author information

Authors and Affiliations

Authors

Contributions

D.X., Z.L. and Z.R. conceived the study. R.Y., and Z.M. performed molecular cloning, protein expression and purification, biochemical and structural experiments with assistance from L.Y., F.W., S.M., M.Y., C. Yan, S.L., J.Y., Li C., D.M., Z.S. and L.W.; Z.L. determined the crystal structure with assistance from L.Y., Z.M., S.F. and Y.S.; S.L. and R.Y. performed genetic assays with the help from C. Yu, Y.L.; Linhai C. and F.N. synthesized 2H3-4BD; D.X., Z.L., Z.R., R.Y., Z.M., L.Y., S.L., J.L., H.D., W.H. and J.C. analysed data with help from all authors; D.X., Z.L., R.Y. and Z.M. wrote the paper with the inputs from all authors.

Corresponding authors

Correspondence to Zihe Rao, Zhiyong Lou or Daoxin Xie.

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The authors declare no competing financial interests.

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Reviewer Information

Nature thanks P. McCourt, K. Snowden and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 D3 orthologues among different plant species and complementation of Arabidopsis max2 by rice D3.

a, Secondary structure elements of D3 in the crystal structure of D3–AtD14 complex are shown on top of Oryza sativa D3 sequence. Identical and conserved residues are highlighted by red and white grounds, respectively. The 19 LRRs are indicated by solid magenta rectangles. The GenBank accession numbers from top to bottom: Oryza sativa D3 (BAD69288), Arabidopsis thaliana MAX2 (NP565979) and Petunia hybrida MAX2 A (AEB97384). b, Branching phenotypes of seven-week-old Col-0, max2-3 and two independent lines (L1 and L2) of max2-3 plants expressing Flag-tagged rice D3 (max2::D3). Scale bar, 1 cm. The images are representative of 20 plants for each genotype. c, The Flag–D3 protein in corresponding plants (b) was detected by anti-Flag antibody (upper panel). Staining of Rubisco large subunit (LSU) served as a loading control (lower panel). Full blots are shown in Supplementary Fig. 1.

Extended Data Figure 2 A proposed mechanism of AtD14-mediated SL hydrolysis.

a, CLIM forms stable contacts with S97 and H247 in the catalytic centre of AtD14. CLIM, the nearby contacting residues and two catalytic residues (S97 and H247) are represented as sticks. The 2FoFc omit map (black mesh) for CLIM is contoured at 1σ and the 2Fo − Fc electron density maps (red mesh) for CLIM, S97 and H247 are contoured at 1σ (left panel) and 1.5σ (right panel), respectively. b, D3 inhibits the release of D-OH during the AtD14-mediated GR24 hydrolysis. Released D-OH in each indicated reaction mixture after 60 min incubation at 37 °C were quantitated by LC-MS/MS. 50 μM GR24, 8 μM AtD14, and/or 15 μM D3 were used. Data are means ± s.d. (n = 6). c, Proposed mechanism of AtD14-mediated GR24 (compound 1) hydrolysis. The reaction begins with the well-accepted nucleophilic attack by S97 in the catalytic triad1,2,24,30. Decomposition of the tetrahedral intermediate I resulted in the enolate form of ABC-OH (2) (which upon protonation gives compound 2) and the S97-bound moiety 3. The alternative direct hydrolysis of GR24 (1) is not supported by the fact that AtD14 S97 mutant is catalytically inactive1. Likewise, direct hydrolysis of the ester bond in S97-bound moiety 3 is an unlikely event. As the electron density shape (Extended Data Fig. 2a) and the C5H5O2 modification (Fig. 1c, d and Extended Data Figs 3 and 4) support covalent bond between H247 and the C5 unit moiety, the transformation from S97-bound moiety 3 to D-OH (6) proceeds via the following cascade: (i) The Nε2 atom of H247 attacks the aldehyde carbonyl of the S97-bound moiety 3 to form the hemiaminal 4, referred to as the covalently linked intermediate molecule (CLIM). The structure of the H247- and S97-linked 4 is consistent with the electron density shape (Extended Data Fig. 2a). (ii) Lactonization of the hemiaminal 4, via a second tetrahedron intermediate (not shown), gives free S97 and H247-bound lactone 5. The structure of 5 is consistent with the C5H5O2 modification of H247 detected by MS/MS (Fig. 1c, d and Extended Data Figs 3 and 4). (iii) Finally, the hydrolysis of H247-linked C5 unit will produce D-OH (6). A more detailed chemistry analysis is shown in the Supplementary Notes. d, Proposed mechanism of AtD14-mediated 4BD hydrolysis, which is similar to that of GR24.

Extended Data Figure 3 Various SL molecules generated the same C5H5O2 modification of AtD14.

a, Chemical structures of rac-GR24 (racemic GR24), 4BD and 5DS with the conserved D-ring. b, GR24-induced C5H5O2 modification of H247 of AtD14 was identified. MS/MS spectra of a triply charged peptide (244-TEGhLPQLSAPAQLAQFLR-262) of AtD14 at mass-to-charge ratio (m/z) 725.0528 corresponding to the mass of the C5H5O2 modification of H247. The AtD14 gel bands of the SEC-separated GR24-induced AtD14–D3 complex were excised for trypsin digestion and MS/MS analysis. Labelled peaks correspond to masses of y and b ions of the modified peptide. Lowercase ‘h’ indicates the modification on H247 by a GR24 hydrolysis intermediate with the molecular formula C5H5O2. c, Size-exclusion chromatography analysis of the 5DS- or 4BD-induced AtD14–D3 interaction using the same method as described in Fig. 1b. d, e, 4BD (d) and 5DS (e) induced the same C5H5O2 modification of H247, which was detected using the in-gel digestion method as described in b.

Extended Data Figure 4 SLs generated the C5H5O2 modification of AtD14 in planta.

a, b, The existence of CLIM in vivo was suggested by successful identification of the same C5H5O2 modification on the AtD14–Flag purified from plants pre-treated with GR24 (a) or 5DS (b). The MS/MS analysis was performed with the same method as mentioned in Extended Data Fig. 3b. c, No modification was observed on the 247th amino acid of AtD14(S97A). The MS/MS spectra of a triply charged peptide 244-TEGHLPQLSAPAQLAQFLR-262 of AtD14(S97A) at mass-to-charge ratio (m/z) 693.0458 corresponding to the mass of unmodified peptide, which were digested from AtD14(S97A) band separated by SDS–PAGE of the mixture containing AtD14(S97A), GR24 and D3–ASK1. d, No modification was observed on the 247th amino acid of AtD14(H247A). The MS/MS spectra of a triply charged peptide 244-TEGALPQLSAPAQLAQFLR-262 of AtD14(H247A) at m/z 671.0434 corresponding to the mass of unmodified peptide, which were digested from AtD14(H247A) band separated by SDS–PAGE of the mixture containing AtD14(H247A), GR24 and D3–ASK1. e, Size-exclusion chromatography analyses showed that the alanine substitution on S97 or H247 of AtD14 disrupted the GR24-induced interaction between the AtD14 and D3–ASK1. The size-exclusion chromatography and SDS–PAGE are performed similarly as described in Fig. 1b.

Extended Data Figure 5 Carba-GR24 and D-OH could neither induce AtD14–D3 interaction nor generate the C5H5O2 modification on AtD14.

a, Pull-down assays using recombinant His6–MAX2 and GST–AtD14 with indicated concentration of carba-GR24 and GR24. Full blots are shown in Supplementary Fig. 1. b, The MS/MS spectra of a triply charged peptide 244-TEGHLPQLSAPAQLAQFLR-262 of AtD14 at m/z of 693.0450 corresponding to the mass of an unmodified peptide, which was digested from the AtD14 bands separated by SDS–PAGE of the mixture containing AtD14, carba-GR24 and D3–ASK1. c, Chemically synthesized D-OH was unable to induce AtD14–D3 interaction. AtD14 was incubated with D3–ASK1 in the presence of 8 mM D-OH at 25 °C for one hour, followed by size-exclusion chromatography analysis as described in Fig. 1b. d, The MS/MS spectra of a triply charged peptide 244-TEGHLPQLSAPAQLAQFLR-262 of AtD14 at m/z of 693.0454 corresponding to the mass of an unmodified peptide, which was digested from the AtD14 bands separated by SDS–PAGE of the mixture containing AtD14, D-OH and D3–ASK1.

Extended Data Figure 6 The MS spectra and chromatograms of the C5H5O2-modified peptide of AtD14.

a, The MS spectra of a bivalently charged peptide (244-TEGhLPQLSAPAQLAQFLR-262) of AtD14 at m/z of 1087.0736 or 1088.5828 corresponding to the mass of C5H5O2 or C5H22H3O2 modification of the peptide, which was isolated from AtD14 in the 4BD or 2H3-labelled 4BD-induced AtD14–D3 complex with in-solution digestion method, respectively. b, The chromatograms of the unmodified (top panel), C5H5O2- (middle panel) or C5H22H3O2-modified (bottom panel) peptide of AtD14 (244-TEGHLPQLSAPAQLAQFLR-262) in the LC-MS/MS analyses. The mass spectra of the C5H5O2- or C5H22H3O2-modified peptide are shown in a.

Extended Data Figure 7 Comparison of different D14 structures.

af, Related to Fig. 2, the characteristic features of two previously reported D3-unbound OsD14 structures, focusing on the shape (a, d), volume (b, e) and details (c, f) of the catalytic centre. (OsD14–TMB, PDB code: 4IHA; OsD14–D-OH, PDB code: 3WIO). g, Sequence alignment and structural annotation of D14 orthologues among different species. Secondary structure elements of AtD14 in the crystal structure of D3–AtD14 complex are displayed on top of the Arabidopsis thaliana D14 sequence. Identical and conserved residues are highlighted by red and white grounds, respectively. The three catalytic residues, S97, D218 and H247, are indicated by magenta dots. The GenBank accession numbers for sequences from top to bottom: Arabidopsis thaliana D14 (Q9SQR3), Oryza sativa D14 (Q10QA5) and Petunia hybrida DAD2 (AFR68698).

Extended Data Figure 8 Structures of D3–ASK1 and mutagenesis analyses of the AtD14–D3 interaction.

a, b, Overall structure of AtD14-bound D3–ASK1 in top view (a) and side view (b). ASK1 and D3 are coloured as pale green and rainbow scheme, respectively. LRRs are labelled. The unusually long loops in LRR16, LRR17 and LRR19 (denoted as loop16, loop17 and loop19) are indicated. The missing part of LRR13 is denoted as a dashed line. c, d, Structural comparison between AtD14-bound D3–ASK1 (magenta) and the apo D3–ASK1 (pale green), with their orientations related to a and b. The indicated loop15, loop16, loop17 and loop19 undergo significant conformational changes during AtD14 binding. e, f, Amino acid substitution of the central residues (P161AtD14, E174AtD14, R177AtD14, D606D3, L644D3 and R702D3) on the interface attenuate interaction between AtD14 and D3–ASK1. GR24 was supplemented in all the reactions. The GR24-induced interactions between D3–ASK1 and wild-type AtD14 or unrelated mutant AtD14(S58A) were used as the control. The size-exclusion chromatography and SDS–PAGE were performed as described in Fig. 1b.

Extended Data Figure 9 Gly158 plays an important role in the SL-induced open-to-closed transition of AtD14 lid.

a, AtD14–Flag protein levels in two independent lines (L1 and L2) of the d14-5 plants with transgenic expression of Flag-tagged AtD14 (d14-5::AtD14, mentioned in Fig. 4b) (upper panel). Staining of Rubisco large subunit (LSU) served as a loading control (lower panel). Full blots are shown in Supplementary Fig. 1. b, AtD14(G158E) was incubated with D3–ASK1 in the presence of GR24, followed by size-exclusion chromatography analysis as described in Fig. 1b. c, AtD14(G158E) maintains the SL-induced interaction with SMXL6. Pull-down assays using recombinant SMXL6–Flag and GST–AtD14 or GST–AtD14(G158E) in the absence or presence of 20 μM GR24. Full blots are shown in Supplementary Fig. 1. d, G158AtD14 participates in the construction of a π-turn structure and the formation of a hydrogen bond with R652D3. The π-turn structure, and structural elements of AtD14 and D3 are coloured as blue, light blue and salmon, respectively. The π-turn residues include A154, W155, V156, H157, G158 and F159. e, During the open (apo AtD14, cyan) to closed (D3-bound AtD14, green) state transition of AtD14, the αT2 and αT3 helices of the lid domain experience pronounced structural and positional alterations (upper panel). In the open state (lower-left panel), the N terminus of αT2 and portions from αT1 and its immediate preceding loop are more flexible than other parts of the AtD14 lid domain, as judged by the higher temperature factors (B-factors) of these regions. In the closed state (lower-right panel), the π-turn structure, with an intrinsic capacity to stop further extension of the αT1 C terminus during the open-to-closed transition of AtD14, contributes to stabilizing proper conformation of the AtD14 lid in the AtD14–D3 complex. Therefore, substitution of Gly158 with a good helix-forming residue Glu would expectedly impair normal conformational changes of the lid which was observed in the AtD14–D3 complex.

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Notes (a detailed deduction of the D-ring-derived intermediates and the reaction process of SL hydrolysis) and Supplementary Figure 1 (the full blot scans). (PDF 504 kb)

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Yao, R., Ming, Z., Yan, L. et al. DWARF14 is a non-canonical hormone receptor for strigolactone. Nature 536, 469–473 (2016). https://doi.org/10.1038/nature19073

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