Extended Data Fig. 9: GRP20 condensates in Arabidopsis and tobacco cells, and HDR is required for flower development and condensate formation.
a, The disorder confidence score along the GRP20 protein predicted by Phyre2. There are two highly disordered regions (60%-100%) in GRP20, one near the N-terminus (residue 15 to 25, a region with positive charge) and highlighted by light green; the other is the C-terminal region (residue 119 to 153, highly disordered region, HDR) and highlighted by light blue. b, Illustration of GRP20 protein structures for various transgenic constructs. c-e, Analyses of different transgenic plants with HDR constructs, as shown in Extended Data Fig. 9b. (c) Comparison of protein expression level in transgenic plants, using Western blot with antibodies against GFP and the same amount of input flower proteins indicated by antibodies against β-Tubulin. The GRP20ΔHDR transgenic plants expressed the GRP20 protein at a level higher than that in the wild-type. (d) Flower phenotypes in transgenic plants as labeled at the top. Yellow letters and numbers at the right top of each panel indicate the organ and corresponding number. P: Petal; S: Stamen. Bar = 1 mm. (e) Floral organ numbers in transgenic plants. Flower counts: ProGRP20::GRP20-YFP grp20, 30; ProGRP20::GRP20ΔHDR-YFP grp20, 30 and grp20-1, 26. Data are presented as mean ± SD. Two-sided Student’s t test. f, The GRP20 condensates in Arabidopsis petal nuclei in complementation lines. The nuclei were stained by DAPI. The condensates are indicated by yellow arrows. BF: Bright field. The bottom right panel is a magnified image of red boxed area in the top left panel. Bar= 5 μm. g, GRP20 condensates in a tobacco leaf cell following transient transformation. The nucleus is also stained by DAPI. Red arrows indicate some of nuclear condensates and yellow arrows indicate some of the condensates in the cytosol. Bar= 5 μm. h, The liquid fluidity of GRP20 condensates tested by photo-bleaching in tobacco cells. The YFP fluorescent signal was recorded from 0 second(s) to 180 s. The YFP condensates were bleached by 100% 514 nm laser from 7 to 11 s after the start of recording. Red arrows mark the positions of strong YFP fluorescence signals, yellow arrows indicate weak YFP signals (following bleaching), and white arrows indicate the positions corresponding to the pre-bleach YFP signals. i, Fluorescence intensity recovery of YFP signals from the experiments shown in Extended Data Fig. 9h. HDR: highly disordered region. The intensity data are shown by mean ± SD from 15, 10 and 20 condensates for GRP20-YFP, GRP20ΔHDR-YFP, and HDR-YFP, respectively. Bar= 10 μm. j, Condensate formation of various GRP20 proteins with/without deletion of HDR, HDR alone, or RBD mutations, respectively, in tobacco leaf epidermal cells. The condensates were detected in GRP20-YFP, HDR-YFP, and GRP20 (RBDm)-YFP, the signal from GRP20ΔHDR-YFP was relatively even, without obvious condensate. Results support the idea that HDR is necessary and sufficient for condensate formation, but RBD is not necessary to condensate formation. The yellow arrow indicates the nucleus, and the white arrow indicates the condensate, respectively. Bar= 5 μm.
