Fig. 8: Amino acid mutations of T345A, Y633C and L654F contribute to functional divergence of BrJMJ18PC and BrJMJ18Par.

(BrJMJ18Par(A345T) is identical to the sequence of BrJMJ18Par except with a substitution at position 345 at which A was replaced with T, which is specific to BrJMJ18PC; the same goes for BrJMJ18Par(C633Y), BrJMJ18Par(F654L), and BrJMJ18Par(C633Y)/(F654L)). BrJMJ18PC-GFP, BrJMJ18Par-GFP, and its variant proteins were transiently overexpressed in tobacco leaves. H3K36me3 and H3K36me2 were detected using anti-H3K36me3 and anti-H3K36me2 antibodies in 35S::BrJMJ18-GFP expressing leaves under NC for 3 days and NC for 2 days following 1-day of HT, respectively. BrJMJ18-GFP proteins were detected with anti-GFP antibodies to confirm equal expression of exogenous genes. Ponceau S staining was used to assess equal loading of total protein extracts. All the western blot experiments were repeated at least three times with similar results. Source data are provided as a Source Data file.