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The F-box protein UFO controls flower development by redirecting the master transcription factor LEAFY to new cis-elements

Nature Plants volume 9, pages 315–329 (2023)Cite this article

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Abstract

In angiosperms, flower development requires the combined action of the transcription factor LEAFY (LFY) and the ubiquitin ligase adaptor F-box protein, UNUSUAL FLORAL ORGANS (UFO), but the molecular mechanism underlying this synergy has remained unknown. Here we show in transient assays and stable transgenic plants that the connection to ubiquitination pathways suggested by the UFO F-box domain is mostly dispensable. On the basis of biochemical and genome-wide studies, we establish that UFO instead acts by forming an active transcriptional complex with LFY at newly discovered regulatory elements. Structural characterization of the LFY–UFO–DNA complex by cryo-electron microscopy further demonstrates that UFO performs this function by directly interacting with both LFY and DNA. Finally, we propose that this complex might have a deep evolutionary origin, largely predating flowering plants. This work reveals a unique mechanism of an F-box protein directly modulating the DNA binding specificity of a master transcription factor.

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Fig. 1: UFO action is largely independent of its F-box domain.
Fig. 2: LFY and UFO together bind a new DNA motif.
Fig. 3: Functional validation of LUBS.
Fig. 4: The LFY K249R substitution disrupts the LFY–UFO synergy.
Fig. 5: Structural characterization of the ASK1–UFO–LFY–DNA complex.
Fig. 6: LFY–UFO interaction is conserved beyond angiosperm species.

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Data availability

The ampDAP-seq data have been deposited at GEO and are publicly available as of the date of publication (GSE204793). The cryo-EM structure determined in this study is deposited in the EM data bank under the reference number EMD-15145. The .pdb file of the model is available in the Supplementary Information. Any additional information required to reanalyse the data reported in this paper is available from the corresponding author upon request. The biological materials generated in this study are available from the corresponding author without restriction. Source data are provided with this paper.

Code availability

All original code has been deposited at GitHub (https://github.com/Bioinfo-LPCV-RDF/LFYUFO_project) and is publicly available as of the date of publication.

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Acknowledgements

We thank A. M. Boisson for preparing the suspension cells, X. Lai for the ampDAP-seq libraries and technical assistance and R. Koes for sharing data and materials. We acknowledge C. Marondedze, G. Vachon, M. Le Masson, C. Berthollet, B. Orlando Marchesano and J. Bourenane-Vieira for help with the experiments. We thank G. Vert, U. Dolde and R. Dumas for discussion. The electron microscopy facility is supported by the Rhône-Alpes Region, the FRM, the FEDER and the GIS-IBISA. This work used the platforms of the Grenoble Instruct-ERIC centre (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology, supported by FRISBI (ANR-10-INBS-0005-02). We thank C. Mas for assistance and access to the biophysical platform. This work was supported by the GRAL Labex financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003), the CEA (PhD fellowship to P.R.) and the ANR-17-CE20-0014-01 Ubiflor project to F.P.

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Author notes

  1. Hicham Chahtane

    Present address: Green Mission Pierre Fabre, Conservatoire Botanique Pierre Fabre, Institut de Recherche Pierre Fabre, Soual, France

Authors and Affiliations

  1. Laboratoire Physiologie Cellulaire et Végétale, IRIG-DBSCI-LPCV, Université Grenoble Alpes, CEA, CNRS, INRAE, Grenoble, France

    Philippe Rieu, Laura Turchi, Emmanuel Thévenon, Hicham Chahtane, Gabrielle Tichtinsky, Jérémy Lucas, Romain Blanc-Mathieu, Chloe Zubieta & François Parcy

  2. Translational Innovation in Medicine and Complexity, Université Grenoble Alpes, CNRS, Grenoble, France

    Laura Turchi

  3. IBS, Université Grenoble Alpes, CNRS, CEA, Grenoble, France

    Eleftherios Zarkadas & Guy Schoehn

  4. EMBL, ISBG, Université Grenoble Alpes, CNRS, CEA, Grenoble, France

    Eleftherios Zarkadas

  5. Structural Biology Group, European Synchrotron Radiation Facility, Grenoble, France

    Max Nanao

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Contributions

F.P. and P.R. designed the project. P.R. performed the plant experiments with assistance from G.T. P.R. and E.T. performed the biochemical experiments with assistance from H.C. on the evolutionary analyses. L.T. performed the bioinformatics analyses with assistance from J.L. and R.B.-M. E.Z. and G.S. performed the cryo-EM experiments, and M.N., E.Z., C.Z. and G.S. analysed the data. P.R. and L.T. assembled the figures. P.R. and F.P. wrote the paper with contributions from L.T. and C.Z.

Corresponding author

Correspondence to François Parcy.

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Nature Plants thanks Nobutoshi Yamaguchi, Aiwu Dong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 UFO has SCF-dependent and independent functions.

a-c, pAP3 activation measured by DLRA in Arabidopsis protoplasts. EV = Empty Vector (pRT104-3xHA). UFOΔFbox corresponds to a deletion of the whole N-terminal part comprising the F-box domain (aa. 1-90), while UFOdelF corresponds to a previously-described internal deletion in the F-box domain (aa. 50-62)20. Data represent averages of independent biological replicates and are presented as mean ± SD, each dot representing one biological replicate (n = 4). One-way ANOVA with Tukey’s multiple comparisons tests. Stars above bars represent a significant statistical difference compared to 3xHA-LFY + EV or 3xHA-LFY-VP16 + EV negative controls (NS: p > 0.05,*: p < 0.05, **: p < 0.01, ***: p < 0.001 and ****: p < 0.0001). d, Western Blot on protein extracts from independent T1 plants from different phenotypic classes described in Fig. 1g (one independent line per lane). 35S::UFO-5xmyc (line 178-#19) and 35S::UFO-3xFLAG (line 177-#6) plants were used as positive controls. Total proteins were extracted from rosette leaves. Note the difference of molecular weight between UFO and UFOΔFbox. Loss-of-function defects are likely due to silencing of both transgene-encoded UFOΔFbox and endogenous UFO. e, Western Blot on protein extracts from F2 plants described in Fig. 1h. Total proteins were extracted from rosette leaves. f, ufo-1 complementation assay with other 35S::UFO and 35S::UFO∆Fbox lines. Rosette leaves (right, scale bar, 1 cm), inflorescence (middle, scale bar 1 mm) and flower (right, scale bar, 0.5 mm) phenotypes are shown. Primary inflorescences were removed to observe rosette phenotype. For each construct, at least 5 plants were analyzed per line. As in Risseeuw et al, our 35S::UFO lines displayed relatively milder phenotypes than the 35S::UFO phenotypes reported by Lee et al.6,20. Note that the 35S::UFO-5xmyc 178-#2 line did not display the serrated leaves phenotype. g, Sequence alignment of UFO N-terminal region. The F-box domain is represented76. In selected species, presented proteins were identified as UFO homologs and their role was confirmed genetically7,11,12,16,77,78,79,80,81,82,83,84. Source data are available in Supplementary Data 4.

Source data

Extended Data Fig. 2 pAP3 DEE LFYBS is not required for LFY-UFO-dependent pAP3 activation.

a, Schematic representation of pAP3. Top row represents WT pAP3 with regulatory regions and cis-elements. Orange triangle represents LFYBS. The second row represents the scores for the best LFYBS obtained by scanning WT pAP3 sequence with LFY PWM68 (the best binding sites correspond to the less negative score values). Other rows represent the different pAP3 versions used in (b) and (c). LFYBS mutation corresponds to the previously described site1m-site2m mutation24. b,c, pAP3 activation with promoter versions described in (a) and indicated effectors. For bar charts, data represent averages of independent biological replicates and are presented as mean ± SD, each dot representing one biological replicate (n = 4). Unpaired t-tests (b,c). (NS: p > 0.05,*: p < 0.05, **: p < 0.01, ***: p < 0.001). Source data are available in Supplementary Data 4.

Extended Data Fig. 3 Analysis of pAP3 activation by LFY-UFO.

a, Description of pAP3. Top line represents WT pAP3 with regulatory regions and cis-elements. Coordinates are relative to AP3 start codon. TSS: Transcription Start Site. Orange triangle represents LFYBS. Other rows show the promoter versions used in (b) and (c). Green rectangles in swapped versions correspond to the same random sequence. b,c, pAP3 LFY-UFO response element mapping with pAP3 versions described in (a) by DLRA in Arabidopsis protoplasts. Data represent averages of independent biological replicates and are presented as mean ± SD, each dot representing one biological replicate (n = 4). One-way ANOVA with Tukey’s multiple comparisons test (c). One-way ANOVA was performed with data from the same effector, and stars represent a statistical difference compared to WT pAP3. Unpaired t-tests (b). (NS: p > 0.05,*: p < 0.05, **: p < 0.01; ***: p < 0.001). d, EMSA with ASK1-UFO, LFY-DBD and LUBS0 DNA probe. Different competitor DNA concentrations were tested as indicated. e, Molecular mass determination for ASK1-UFO-LFY-DBD in complex with LUBS0 DNA by SEC-MALLS (top). Elution profiles correspond to absorbance at 280 nm and 260 nm (left ordinate axis, A.U: Arbitrary Unit). The black line shows the molecular mass distribution (right ordinate axis). A mass of 102 ± 3.3 kDa was found for this ASK1-UFO-LFY-DBD-LUBS0 complex, consistent with one copy of each protein per DNA molecule (theoretical mass of 108 kDa). Coomassie-stained SDS-PAGE gel of the different SEC-MALLS fractions (bottom). Each lane corresponds to a 0.5 mL fraction. Molecular weights of the protein standards are indicated (BioRad Precision Plus). Faint bands above UFO likely correspond to contaminants. f, EMSA with ASK1-UFO, LFY-DBD and indicated DNA probes. Sequences with coordinates relative to AP3 start codon (left). Red letters indicate mutated bases. Bars under sequences represent the regions required for ASK1-UFO-LFY-DBD binding. EMSA with described DNA probes (right). Each DNA probe was mixed with the same ASK1-UFO-LFY-DBD protein mix. Note that the LUBS0 mutation also reduced pAP3 activation in protoplasts (Fig. 2b). Source data are available in Supplementary Data 4.

Source data

Extended Data Fig. 4 Genome-wide analysis of LFY-UFO binding.

a, Western Blot after DNA elution during ampDAP-seq experiment. After DNA elution, 20 µL of 1X SDS-PAGE Protein Sample Buffer was added to the remaining beads to run WB. Each lane represents one replicate. b, Assessment of experimental reproducibility of ampDAP-seq experiment through the comparison of replicates datasets 2 by 2. c, Effect of the LFY KARA mutation (K303A-R233A)51 on pAP3 activation in Arabidopsis protoplasts. Data represent averages of independent biological replicates and are presented as mean ± SD, each dot representing one biological replicate (n = 4). Unpaired t-tests (**: p < 0.01; ****: p < 0.0001). d, The LFY KARA mutation (K303A-R233A) does not disrupt LFY-UFO interaction in Yeast-Two-Hybrid (Y2H). EV = Empty Vector. LFY-40 is a LFY version lacking the first 40 aa and better tolerated by yeast cells. Values correspond to the different dilutions (OD = 7, 0.7 and 0.07). Top picture corresponds to the non-selective plate lacking Leucine and Tryptophan (SD -L-W), and bottom picture to the selective plate lacking Leucine, Tryptophan, Histidine and Adenine (SD -L-W-A-H). Pictures were taken at day + 4. e, Receiver operating characteristics (ROC) curves for mLUBS, dLUBS and LFY using the top 20% high-CFC LFY-UFO-specific peaks. Area under the curve (AUC) values are shown. TPR: True Positive Rate, FPR: False Positive Rate. f, Score distribution of LFY PWM with dependencies68 within dLUBS (best site on 20% most LFY-UFO-specific genomic regions, high CFC, n = 3843 genomic regions) and in canonical LFYBS (best site on 20% most LFY-specific genomic regions, low CFC, n = 3843 genomic regions). Best sites were selected within ±25 bp around the peak maximum. Wilcoxon rank sum test (****: p < 0.0001). Median (solid line), interquartile range (box edges), ±1.5 × interquartile range (whiskers) and outliers (black dot) are shown. g, De novo identification of URM from LFY ChIP-seq data25. Motifs identified at a fixed distance from LFY canonical binding sites in 298 regions harboring high LFY ChIP-seq to LFY ampDAP-seq coverage ratio. The text above each motif gives the motif’s start position relative to the canonical LFYBS, its length and the number of sites used to build the motif. h, EMSA with mLUBS and dLUBS highest score sequences. 6xHis-LFY-DBD is recombinant. UFO* refers to either recombinant ASK1-UFO-3xFLAG complex (top gel) or in vitro produced UFO-3xFLAG (bottom gel). Drawings represent the different types of complexes involving LFY-DBD (pale blue) and ASK1-UFO (red) on DNA. LFY-DBD binds as a monomer as previously reported29. The fact that in vitro produced UFO-3xFLAG shifts DNA in the presence of LFY indicates that ASK1 is not required for the UFO-LFY-DNA complex formation in vitro. i, EMSA with DNA probes corresponding to pAP1 and pAP3 DEE LFYBS and indicated proteins. Note that probes used here have the same length as those used to study LUBS. Source data are available in Supplementary Data 4.

Source data

Extended Data Fig. 5 pAP3 LUBS are required for LFY-UFO-dependent activation.

a, EMSA with indicated probes and proteins. LUBS3 is the third highest-score pAP3 LUBS. Because LUBS0 is bound with a lower affinity by LFY-UFO compared to LUBS1 and LUBS2, we then focused on LUBS1 and LUBS2. b, EMSA with pAP3 LUBS1 and LUBS2 DNA probes and indicated proteins. LFYH383A-R386A (LFYHARA) is a LFY mutated version affected in its ability to dimerize29,51. Note the absence of the complex with a slower mobility on LUBS1 with LFYHARA. c, EMSA with pAP3 LUBS1 and LUBS2 DNA probes and indicated proteins. LFY* refers to either in vitro-produced 5xmyc-LFY (top) or recombinant 6xHis-LFY-DBD (bottom). Note the difference of complex size between UFO and UFOΔFbox. d, Same as in (c) except that UFO-3xFLAG and UFO∆Fbox-3xFLAG were produced in vitro. Note that in vitro produced UFO-3xFLAG and UFO∆Fbox-3xFLAG behave similarly as recombinant UFO versions. e, EMSA with indicated proteins and DNA probes corresponding to pAP3 LUBS1 (left) and LUBS2 (right), WT or with URM mutated. f, Promoter activation measured by DLRA in Arabidopsis protoplasts with indicated effectors. Different promoter versions were tested as indicated under x-axis. Either 2 bp (high-informative CA) or 6 bp (whole URM) of pAP3 LUBS1 and LUBS2 URM were mutated. Data represent averages of independent biological replicates and are presented as mean ± SD, each dot representing one biological replicate (n = 4). One-way ANOVA with Tukey’s multiple comparisons tests. One-way ANOVA were performed with data from the same effector and stars represent a statistical difference compared to WT pAP3 promoter. (NS: p > 0.05,*: p < 0.05, **: p < 0.01, ***: p < 0.001 and ****: p < 0.0001). g, In vivo analysis of pAP3LUBS1-2m::GUS fusions. Same as in Fig. 3d, except that staining incubation time was increased to 17 h (4 h incubation in Fig. 3d). Representative pictures are shown (top scale bar, 100 µm, bottom scale bar, 50 µm). The faint AP3 pattern suggests that other LUBS (such as LUBS0) may take over but less efficiently. Note that with this staining incubation time, all plants expressing pAP3::GUS showed a highly saturated staining. Source data are available in Supplementary Data 4.

Source data

Extended Data Fig. 6 pRBE LUBS is required for LFY-UFO-dependent activation.

a, IGB view of pRBE showing LFY ChIP-seq in inflorescences (light blue)25 or seedlings (dark blue)26, LFY-UFO ampDAP-seq (yellow), LFY ampDAP-seq (pink)48, numbers indicate read number range (top). Identification of LUBS in pRBE (bottom). Predicted binding sites using dLUBS and mLUBS models from Fig. 2e and LFY PWM with dependencies68, y-axis represents score values (bottom). The best binding sites correspond to the less negative score values. Studied LUBS is indicated with a purple square. b, EMSA with probes corresponding to pRBE LUBS, WT or with URM mutated. c, pRBE activation in Arabidopsis protoplasts. Effect of mutations (underlined) in URM (red) and in LFYBS (blue) bases of pRBE LUBS were assayed. Data represent averages of independent biological replicates and are presented as mean ± SD, each dot representing one biological replicate (n = 4). One-way ANOVA with Tukey’s multiple comparisons test. One-way ANOVA were performed with data from the same effector, and stars represent a statistical difference compared to WT promoters (****: p < 0.0001). d, In vivo analysis of pRBE::GUS fusions. The percentage of transgenic lines with RBE pattern, unusual pattern or absence of staining was scored (top; χ² test, **: p < 0.01). n = number of independent lines. Unusual pattern refers to staining in unexpected tissues, each pattern seen in a single line. Representative pictures of plants with no staining (bottom left) and a RBE pattern (bottom right) are shown (scale bar, 50 µm). e, In vivo analysis of pRBE::GUS fusions. Same as in (d), with another view showing staining in the four petal primordia (scale bar, 50 µm). Source data are available in Supplementary Data 4.

Source data

Extended Data Fig. 7 LFY and UFO likely regulate other genes in Arabidopsis.

a, List of candidate LFY-UFO target genes selected as i) present in regions specifically bound by LFY-UFO in ampDAP-seq (high CFC) ii) bound in vivo in LFY ChIP-seq experiments (A25; B26; C68; D73) and iii) deregulated in ufo inflorescences70. b, IGB view of PISTILLATA promoter region showing LFY ChIP-seq in inflorescences (light blue)25 or seedlings (dark blue)26, LFY-UFO ampDAP-seq (yellow), LFY ampDAP-seq (pink)48, numbers indicate read number range (top). Predicted binding sites using the dLUBS, mLUBS models from Fig. 2e and LFY PWM with dependencies68, y-axis represents score values (bottom). c, IGB view of selected genes showing LFY ChIP-seq in inflorescences (light blue)25, LFY-UFO ampDAP-seq (yellow), LFY ampDAP-seq (pink)48, numbers indicate read number range. Genes in red are deregulated in ufo inflorescences70. ChIP-seq peaks better explained by LFY-UFO than by LFY alone are shaded in grey. Source data are available in Supplementary Data 4.

Extended Data Fig. 8 The LFY K249 is essential for LFY-UFO-LUBS complex formation.

a, Structure of LFY-DBD29. Residues were colored by conservation using Consurf with default parameters85. K249 residues on each LFY monomer are represented as sticks and indicated with arrows. Note that the K249-containing loop is highly conserved. b,c, Promoter activation measured by DLRA in Arabidopsis protoplasts with indicated effectors (right). EV = Empty Vector (pRT104-3xHA). Tested promoters are indicated below each graph. Note that for 3xHA-LFY + UFO-3xFLAG on pAG only n = 3 biological replicates are shown. Data represent averages of independent biological replicates and are presented as mean ± SD, each dot representing one biological replicate (n = 4 unless specified). One-way ANOVA with Tukey’s multiple comparisons tests (b) or Welch’s ANOVA with Games-Howell post-hoc test (c). In (c), stars above bars represent a statistical difference compared to GFP. Other comparisons are indicated with brackets. (NS: p > 0.05,*: p < 0.05, **: p < 0.01, ***: p < 0.001 and ****: p < 0.0001). d, Effect of the LFYK249R mutation on LFY-UFO interaction in Y2H. EV = Empty Vector. LFY-40 is a LFY version lacking the first 40 aa and better tolerated by yeast cells. Values correspond to the different dilutions (OD = 7, 0.7 and 0.07). Top picture corresponds to the non-selective plate lacking Leucine and Tryptophan (SD -L-W), and bottom picture corresponds to the selective plate lacking Leucine, Tryptophan, Histidine and Adenine (SD -L-W-A-H). Pictures were taken at day + 4. e, EMSA with DNA probes corresponding to pAP3 DEE LFYBS and pAP3 LUBS1 and indicated proteins. pAP3 DEE LFYBS DNA probe was used as a control for binding on canonical LFYBS. f, WB after DNA elution during ampDAP-seq experiment. After DNA elution, 20 µL of 1X SDS-PAGE Protein Sample Buffer was added to the remaining beads to run WB. Each lane represents one replicate. g, Reproducibility of ampDAP-seq experiments with LFYK249R (left) and LFYK249R-UFO (right) through the comparison of replicates datasets 2 by 2. h, Comparison of peak coverage in LFYK249R (y-axis, this study) and LFY (x-axis)48 ampDAP-seq experiments. i, Integrated Genome Browser (IGB) view of pAP3 showing LFY ChIP-seq in inflorescences (light blue)25 or seedlings (dark blue)26, LFY-UFO ampDAP-seq (yellow; this study), LFY ampDAP-seq (pink)48 and LFYK249R ampDAP-seq (purple; this study). Numbers indicate read number range. j, Pictures of WT and representative transgenic plants expressing 35S::LFY or 35S::LFYK249R (scale bar, 1 cm). The white arrows indicate ectopic rosette flowers. 35S::LFY was obtained previously26. 42 T1 plants expressing 35S::LFYK249R were analyzed; the percentage of plants with a LFY overexpressing phenotype is comparable to the one obtained with 35S::LFY26. Source data are available in Supplementary Data 4.

Source data

Extended Data Fig. 9 UFO binds DNA and LFY DBD.

a, A representative micrograph of the ASK1-UFO-LFY-DNA complex in vitreous ice (scale bar, 20 nm). b, Selected 2D class averages of the particles submitted to ab initio reconstruction and heterogeneous refinement for 3D classification. c, Intermediate reconstructions of the 3D classes after heterogeneous refinement. d, Final reconstructions of ASK1-UFO-LFY-DNA complexes (involving either a LFY-DBD monomer (pink) or a LFY-DBD dimer (gray)) after Non-Uniform refinement. e, Unprocessed AlphaFold2 model for ASK1 (top, purple; uniprot ID, Q39255), UFO (middle, red; uniprot ID, Q39090) and the LFY-DBD dimer/DNA crystallographic structure (bottom, pale and dark blue for the LFY-DBD dimer and green for the DNA; PDB, 2VY1). f, Cryo-EM density map color-coded by fitted molecule. Note the kink on DNA induced by the presence of UFO. g, Heat map of the angular distribution of particle projections contributing for the final reconstruction of the complete ASK1-UFO-LFY-DNA complex (with a LFY-DBD dimer). h, Gold-standard Fourier shell correlation (FSC) curves. The dotted line represents the 0.143 FSC threshold, which indicates a nominal resolution of 6.4 Å for the unmasked (red) and 4.3 Å for the masked (blue) reconstruction. i, View of the post-processed map of the complete ASK1-UFO-LFY-DNA complex, colored according to the local resolution.

Supplementary information

Reporting Summary

Supplementary Data 1

List of plasmids used in this study.

Supplementary Data 2

List of oligonucleotides used in this study.

Supplementary Data 3

Cryo-EM data collection and refinement statistics.

Supplementary Data 4

Statistical source data for Figs. 1–6 and Extended Data Figs. 1–8.

Supplementary File 1

Model for the LFY–ASK1–UFO–DNA complex.

Source data

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Rieu, P., Turchi, L., Thévenon, E. et al. The F-box protein UFO controls flower development by redirecting the master transcription factor LEAFY to new cis-elements. Nat. Plants 9, 315–329 (2023). https://doi.org/10.1038/s41477-022-01336-2

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