Journal: Nature Communications

Article Title: Shade-induced RTFL/DVL peptides negatively regulate the shade response by directly interacting with BSKs in Arabidopsis

doi: 10.1038/s41467-023-42618-3

Figure Lengend Snippet: a The expression of RTFL18 in different tissues was determined on the basis of luciferase signal intensity in pRTFL18::Luc transgenic lines. I, III, seedlings were grown under white light for 8 (I) or 11 (III) days; II, IV seedlings were grown under white light for 3 days and transferred to low R:FR and grown for 5 (II) or 8 (IV) days; V, rosette leaves from plants grown under long day conditions for 4 weeks; VI, cauline leaves, VII, fruits, and VIII, flowers from plants grown under long day conditions for 6 weeks. b The relative expression of RTFL18 in different tissues under WL and low R:FR conditions was measured by RT‒qPCR. The expression in the roots under WL was standardized to be “1”. Data are presented as mean values +/− SD ( n = 3, n refers to biological replicates). Letters indicate significant differences between mean values (one-way ANOVA, P < 0.01), and groups with the same letters are not significantly different. c The protein levels of RTFL18 under WL and low R:FR conditions were measured by western blotting. Whole seedlings were used for experiments. Western blots were probed with anti-Flag antibody. The levels of β-tubulin indicated the loading control. d Subcellular localization of GFP-RTFL18 was measured by transient expression assay in Nicotiana benthamiana leaves. e Subcellular localization of Flag-RTFL18 was measured by immunolocalization assay in Arabidopsis seedlings. Flag-RTFL18 (green) was labeled by immunolocalization using anti-Flag antibodies and observed with a confocal laser scanning microscope. The membrane was stained with FM4–64 (red). GFP, GFP signal; FM4–64, FM4–64 staining; Flag, Flag signal; Bright, bright field; Merge, merged image of the GFP/Flag signal with the FM4–64 signal in a bright field. f Subcellular localization of Flag-RTFL18 was measured by western blotting. Western blots were probed with anti-Flag, anti-H + −ATPase (Plasma membrane marker), anti-Rbcl, and anti-H3 antibodies. Scale bars represent 1 cm ( a ), and 20 μm ( d , e ). In c – f , each experiment was repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: In Fig. , proteins in different cellular fractions were extracted from 7-day-old 35 S::Flag-RTFL18 transgenic seedlings using a Minute TM Plasma Membrane Protein isolation kit (MobiTec, SM-005-p-INV) and subjected to SDS-PAGE electrophoresis and immunoblot analysis; PM H + -ATPase was detected using Anti-H + -ATPase antibody (PHYTOAB, PHY2285A) nuclear H3 was detected using anti-H3 antibody (Abmart, P30266M), and cytoplasm Rbcl was detected using anti-Rbcl antibody (Agrisera, AS03037).

Techniques: Expressing, Luciferase, Transgenic Assay, Western Blot, Control, Labeling, Laser-Scanning Microscopy, Membrane, Staining, Clinical Proteomics, Marker