Fig. 7: Arginine methylation of R10 promotes its ubiquitination.

a Protein levels of R10 in WT and osprmt6b-1 mutant treated with 0 μM or 10 μM ABA for 3 and 6 hours. HSP82, loading control. The WT and osprmt6b-1 seedings were simultaneously treated with 1 mM CHX (n = three independent experiments). b The mRNA levels of OsPRMT6b in rice seedlings treated with 0 μM or 1 μM ABA. Samples were harvested at 0, 1, 3, 6, 12, and 24 h after treatments. CK served as the control (n = two independent experiments). Error bars, standard deviation (All above P values were calculated by two-tailed t-test. c Levels of OsPRMT6b protein in WT and osprmt6b-1 mutant seedlings treated with 0 μM or 1 μM ABA. The membrane was probed with OsPRMT6b and HSP82 antibodies (n = three independent experiments). (d) Western blot assay showing the protein levels of R10-GFP, R10^3K-GFP, and R10^3F-GFP in 14-d-old R10-GFP, R10^3K-GFP, and R10^3F-GFP overexpressing lines. Samples for western blot analysis occurred at the indicated time points. HSP82, loading control. e Ubiquitination assays of R10, R10^3K, and R10^3F proteins incubated with WT and osprmt6b-1 plant total protein extractions. The ubiquitination of R10, R10^3K or R10^3F was detected by Anti-Ub antibody (n = three independent experiments). Anti-His, the protein loading control. Ub, Ubiquitination. f Ubiquitination and methylation of R10-GFP protein in rice protoplasts of WT and osprmt6b-1 mutant. Total protein of protoplasts was extracted, proteins separated using SDS-PAGE, and levels of ubiquitination and methylation detected using respective antibodies. Anti-GFP was used as a loading control (n = three independent experiments). g In vivo co-immunoprecipitation assays of TE with R10, R10^3F, and R10^3K in rice. The R10, R10^3F, and R10^3K proteins were enriched by GFP beads, and an equal amount of these enriched proteins was incubated with the total protein of His-TE overexpressing transgenic line (n = three independent experiments). Numbers below the top band show relative protein levels in a, c–g. Source data are provided as a Source Data file in a–g.