Fig. 7: MCA–IIs directly interact with PUX10 in cells.

a Confocal micrographs of lipid droplets stained with LipidTOX in the hypocotyl region of embryos after 2 days stratification of a line co-expressing RPS5apro:MCA-II-a-tagRFP and PUX10pro:PUX10-GFP. In the insets denoted with “a”, orange arrowheads show a lack of colocalization between MCA-II-a/PUX10 with LipidTOX; “(b)”, yellow arrowheads show the MCA-II-a/PUX10 colocalization with LipidTOX; “(c)”, same as in “(a)”, showing that lack of colocalization with relatively larger droplets. Scale bars, 10 μm. The micrographs on the bottom show a surface Z-axis of cells, for better visualization of the PUX10 droplets. The yellow arrowheads show colocalized PUX10/MCA-II-a, while the orange arrowhead shows a lack of colocalization (note the increased signal intensity of PUX10 in this instance). The Pearson correlation coefficient (r) represents the colocalization between GFP and tagRFP signals in droplets. Scale bars, 10 μm (N > 5, n = 1 replicate with ≥10 seedlings). b Representative confocal micrographs (lower) of PLA signal (PLA foci denoted with magenta) indicating the interaction between HF-MCA-II-a-tagRFP (HF: 6xHis-3xFLAG tag) with PUX10-GFP (α-FLAG and α-GFP) from epidermal cells of meristematic cells from root. Yellow arrowheads denote detectable PUX10/MCA-II-a signals, while the orange arrowhead denotes submicroscopic PUX10/MCA-II-a signals that produce PLA (i.e., interactions outside droplets). Scale bar (in “PLA” for visibility), 10 μm. Lower chart: corresponding quantification of PLA-positive foci. P-values were calculated by one-way ANOVA (N = 3, n = 1 replicate with multiple cells). c The “sensitized emission” FRET (FRET-SE) approach, where the emission spectrum of the donor (1) overlaps with the excitation spectrum of the acceptor (2), and if the distance between the two molecules is sufficiently short (i.e., connoting association), energy is transferred (3). d Confocal micrograph showing FRET-SE intensity between PUX10-GFP and MCA-II-a-tagRFP from root epidermal cells. Scale bar, 5 μm. Bottom graph: FRET-SE between PUX10-GFP and MCA-II-a-tagRFP in the absence (mock) or presence of 50 μM MG132. GFP represents the negative control (free GFP; lines co-expressing GFP with MCA-II-a-tagRFP). P-values were calculated by ordinary one-way ANOVA (N = 2, n = 2 roots ≥ 7 cells 5 DPG). Source data are provided as a Source Data file.