Fig. 1: SPL/NZZ directly interacts with MADS-domain TFs. | Nature Communications

Fig. 1: SPL/NZZ directly interacts with MADS-domain TFs.

From: SPOROCYTELESS/NOZZLE cooperates with MADS-domain transcription factors to regulate an auxin-dependent network controlling Megaspore-Mother-Cell differentiation

Fig. 1

A Vulcano plot showing, with purple dots, the TFs statistically enriched in the SPL/NZZ Co-IP/MS dataset. MADS-domain TFs, SPL/NZZ and TCP22 have been highlighted with labels. The experiment has been performed using three independent biological replicates of SPL/NZZ:GFP AP1:GR ap1cal inflorescences at 9 DAI. Proteins with a positive enrichment (log2 Intensity difference > 0) and FDR ≤ 0.05 were considered significant. The full list of SPL/NZZ interactors identified by Co-IP/MS can be found in Supplementary Data 1. B SPL/NZZ-MADS-domain TFs (AG, STK and SEP3) direct interaction by yeast-two-hybrid assay. Interactions are tested on media depleted of histidine (-W -L -H), or histidine and adenine (-W -L -H -A). For each interaction on the different media, yeast has been spotted at four different concentrations according to a serial dilution (OD 0.5; 1:10; 1:100; 1:1000). C SPL/NZZ form complexes with STK, SEP3 and AG by yeast-three-hybrid assays. Interactions are tested on a medium depleted of histidine (-W -L -H -A). For each interaction, yeast has been spotted at four different concentrations according to a serial dilution (OD 0.5; 1:10; 1:100; 1:1000). D–I SPL/NZZ interaction with SEP3 and STK tested by FRET-FLIM. D GFP fluorescence imaged from a nucleus expressing SEP3Δ-GFP. E GFP and mCHERRY fluorescence imaged from a nucleus expressing SEP3Δ-GFP+SPLΔ-mCHERRY. F GFP and mCHERRY fluorescence imaged from a nucleus expressing SEP3Δ-GFP-mCHERRY. G GFP fluorescence imaged from a nucleus expressing STK-GFP. H GFP and mCHERRY fluorescence imaged from a nucleus expressing STK-GFP+SPLΔ-mCHERRY. I Box plot representing the GFP lifetime of nuclei expressing SEP3Δ-GFP, SEP3Δ-GFP+SPL/NZZΔ-mCHERRY, SEP3Δ-GFP-mCHERRY, STK-GFP and STK-GFP+SPL/NZZΔ-mCHERRY, as calculated by FILM combined with a phasor plot to distinguish different lifetime populations. Measurements have been performed on 5, 5, 5, 11 and 5 different nuclei expressing SEP3Δ-GFP, SEP3Δ-GFP+SPL/NZZΔ-mCHERRY, SEP3Δ-GFP-mCHERRY, STK-GFP and STK-GFP+SPL/NZZΔ-mCHERRY, respectively. Source data are provided as a Source Data file. Asterisks over boxes represent the statistical significance as determined by student’s t-test, two-sided distribution, homoscedastic, confronting SEP3Δ-GFP with SEP3Δ-GFP+SPL/NZZΔ-mCHERRY and SEP3Δ-GFP-mCHERRY, or STK-GFP with STK-GFP+SPL/NZZΔ-mCHERRY. **p ≤ 0.01; ***p ≤ 0.001. Exact P values for each comparison can be found in the Source Data file. Box-plots elements correspond to: centre line = median; X = average; box limits = interquartile range; whiskers = lowest and highest values in the 1.5 interquartile range. Single measures are represented with dots in the boxes. Scale Bars: 10 µm.

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