Abstract
Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/β hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.
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Change history
13 January 2016
Nature 504, 406–410 (2013); doi: 10.1038/nature12878 In this Article, the bottom panel of Fig. 3c was assembled from processed digital images of separate western blot autoradiographs to show approximately equal amounts of MBP–D3 protein input in the presence or absence of GR24 treatment. During figure preparation, a single MBP–D3 lane was duplicated in error to create the third and fourth lanes.
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Acknowledgements
We thank T. Omura for providing the d53 mutant, and Z.-Y. Wang and C. Lin for reading of the manuscript. We also thank X. Li for providing the d3 mutant, W. Liu for providing the htd2 mutant and I. Takamure for providing the d10, d17 and d27 mutants. This work was supported by the National Transgenic Science and Technology Program (2011ZX08009-003), 863 National High-tech R&D Program of China (2012AA101100, 2012AA10A301), Jiangsu Province 333 Program (BRA2012126) and National Natural Science Foundation of China (90917017). N.Z. is a Howard Hughes Medical Institute investigator and is supported by the National Science Foundation.
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F.Z., L.Z., X.L., Z.C. and H.Z. performed genetic analysis and mapping of D53; Q.L., Y.R., N.S., F.W. and H.M. performed the protein interaction experiments; X.Z., X.G. and L.J. performed the genetic transformation; C.W., Jiulin.W., Chao.Y., Jie.W. and C.L. performed the construction and identification of double mutants; K.U., S.I., and T.A. performed the synthesis of d6-5DS; W.C., Jinfang.C., and Cunyu.Y. performed the determination of SLs; K.Z., Y.R. and L.G. performed the degradation experiments; F.Z., H.G. and W.D. performed the Y2H and qRT–PCR experiments; Jun.C., D.W., J.T. and W.M. performed the phenotypic analysis and painted the model picture. Jianmin.W., N.Z. and H.W. directed the project and wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Phenotypes of d53 mutant.
a, Comparison of crown root growth in wild-type (WT) and d53 mutant. DAG, days after germination. Each value represents the mean ± s.d. of 25 seedlings. b, Root phenotype of 7-week-old wild type and d53 at the tillering stage. Red dots indicate the main culms. c, Comparison of different types of tillers between wild type and d53 at the heading stage. Pt, primary tillers; St, secondary tillers; Tt, tertiary tillers; Qt, quaternary tillers. Each value represents the mean ± s.d. of 20 seedlings. d, Morphology comparison of tiller buds at the second node between wild type and d53. White arrows and arrowheads indicate the tiller buds and the second nodes, respectively. e, Transverse sections of the first internode of wild type and d53. f, Number of vascular bundles (VB) calculated from transverse sections of the first internode of wild type and d53. SVB, small vascular bundle; LVB, large vascular bundle. Data are means ± s.d. (n = 10). g, Longitudinal sections of the first internode of wild type and d53. h, Comparison of parenchyma (PC) cell length in first internode and root between wild type and d53. Data are means ± s.d. (n = 10). Differences with respect to the wild type that were found to be significant in a t-test are indicated with asterisks (*P < 0.05; **P < 0.01; NS, not significant). Scale bars, 10 cm (b), 2 cm (d), 100 μm (e, g).
Extended Data Figure 2 d53 mutation behaves in a semi-dominant manner.
a, b, Comparison of wild type, heterozygous (F1) and homozygous d53 plants at the heading stage (a), flag leaf (b), cross section of the first internode (c), panicle (d), plant height (e), tiller number (f) and diameter of the third internode (g). Scale bars, 20 cm (a), 5 cm (b, d) and 500 μm (c). For e–g, each value represents the mean ± s.d. (n = 25). h, Segregation of F2 progeny from a self-pollinated F1 plant (d53 × Norin 8).
Extended Data Figure 3 d53 is insensitive to GR24 treatment and confers enhanced tillering-promoting activity.
a, Response of 5-week-old wild type, d53, d14 and d27 to the application of 1 μM GR24. b, Tiller bud length of 2-week-old wild type, d53, d14 and d27 seedling treated with (+) or without (−) 1 μM GR24. Data are means + s.d. (n = 10). ND, not detected. c, Numbers of tillers showing outgrowth (>2 mm) for 5-week-old wild type, d53, d14 and d27 plants treated with (+) or without (−) 1 μM GR24. Data are means ± s.d. (n = 10). Asterisks in c denote significant differences between treated and untreated samples within the same genotype (two tailed Mann–Whitney U test, P < 0.01; NS, not significant). d, D53 RNAi transgenic plants exhibit reduced tillering in the d53 mutant background. d53Vec., d53 transformed with the pCUbi1390-ΔFAD2 control. e, Tiller number of RNAi transgenic lines in d at the tillering stage. Each value represents the mean ± s.d. of six plants (T1 generation). L4, L10 and L11 represent three independent lines. The t-test analysis indicated a significant difference (compared with vector control, **P < 0.01). Scale bars, 20 cm (a), 10 cm (d).
Extended Data Figure 4 Phylogenetic analysis of D53 protein.
Using the D53 protein sequence as the query in tBLASTn searches, homologues were identified from different organisms with a permissive cutoff E value of 1 × 10−3. The sequences chosen from representative genomes were aligned and used to generate the neighbour-joining phylogenetic tree with 1,000 bootstrap replicates. The clade names were given on the basis of known sequences in each clade, which is supported by a bootstrap value > 85.
Extended Data Figure 5 Multiple sequence alignment of the deduced amino acid sequence of D53 with its homologues.
D53 protein is predicted to contain an N-terminal domain, a D1 ATPase domain, an M domain and a D2 ATPase domain (http://toolkit.tuebingen.mpg.de/hhpred). The beginning and ending sites of each domain are indicated above the sequences. The predicted Walker A (P-loop) and Walker B motifs are shown in red boxes in the D1 domain and green boxes in the D2 domain, respectively. Note that the deletion of five amino acids in the D2 domain of d53 protein overlaps with the GYVG loop in ClpC. The conserved putative EAR motif in D53 and ClpP-binding loop in ClpC are also shown. The sequences used for alignment are D53 (Oryza sativa, LOC_Os11g01330), D53-like (Oryza sativa, LOC_Os12g01360), SMXL6 (Arabidopsis, At1g07200), SMXL7 (Arabidopsis, At2g29970) and ClpC (Bacillus subtilis, GI: 16077154).
Extended Data Figure 6 Histochemical staining of the pD53::GUS reporter gene and subcellular localization of D53 protein.
a–h, Histochemical staining of young root (a), shoot (b), leaf (c), leaf sheath (d), panicle (e), transverse section of the leaf sheath (f), stem (g) and node (h). Scale bars, 1 mm (a, b, c, d, f, h), 1 cm (e), 100 μm (g). i–l, Subcellular localization of D53–GFP fusion protein in rice protoplast cells. A nuclear marker protein, OsMADS3, fused with mCherry, was used as a positive control. Scale bars, 5 μm. m–p, Confocal scanning images showing nuclear localization of the D53–GFP fusion protein in transgenic root cells. Scale bars, 100 μm.
Extended Data Figure 7 Mapping of the D14-binding domain of D53.
a, Schematic structure of the D53 protein. Numbers indicate amino-acid (aa) residues. b, Y2H analysis showing interaction between full-length and various domain deletion variants of D53 with D14 in the presence or absence of 5 μM GR24. −LT, control medium (SD −Leu/−Trp); −LTHA, selective medium (SD −Leu/−Trp/−His/−Ade).
Extended Data Figure 8 GR24 promotes D53 protein degradation.
a, Confocal scanning images showing that AEBSF, pepstatin A and leupetin are not effective in blocking D53–GFP fusion protein degradation in transgenic seedlings treated with 5 μM GR24. Scale bars, 100 μm. b, Degradation of D53–GFP fusion protein but not D3–GFP or D14–GFP fusion proteins expressed in rice protoplasts, in the presence of 5 μM GR24. Pre-treatment with 40 μM MG132 for 1 h before addition of GR24 effectively blocks D53–GFP degradation. c, D53–GFP is degraded in the d53 mutant protoplasts in the presence of GR24, but not in d3 or d14 protoplasts. For b and c, each figure represents at least 50 cells observed. Scale bars, 10 μm.
Extended Data Figure 9 D53-RNAi transgenic lines in d3 and d14 backgrounds.
a, Comparison of plant height, diameter of the third internode and tiller number between wild type, d53, d14, d3 and their double mutants. Values are mean ± s.d. (n = 10). b, c, qRT–PCR assay (b) and western blot analysis (c) showing that the endogenous level of D53 messenger RNAs and proteins are downregulated in three representative D53-RNAi lines in d53, d14 and d3 mutant backgrounds, compared to wild-type control. Data are means ± s.d. (n = 3); significant difference determined by t-test (**P < 0.01). Anti-HSP82 was used as a loading control. d, Tiller number of representative D53-RNAi transgenic lines in d3 and d14 mutant backgrounds at the heading stage. Each value represents the mean ± s.d. of six plants. L2, L6 and L11 represent three independent lines in d3 background, and L1, L4, L6, L9 and L10 represent five independent lines in d14 background. Akumuro (A) and Norin 8 (N) are the wild-type varieties correspond to d3 and d14 mutants, respectively. d3Vec. and d14Vec. transgenic lines were used as the controls. Asterisks represent significant difference compared with vector control determined by the t-test at *P < 0.05 and **P < 0.01, respectively.
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Zhou, F., Lin, Q., Zhu, L. et al. D14–SCFD3-dependent degradation of D53 regulates strigolactone signalling. Nature 504, 406–410 (2013). https://doi.org/10.1038/nature12878
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DOI: https://doi.org/10.1038/nature12878
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