Optimized Western Blotting Exploration of H3K27 Methylation Changes under Waterlogging in Barley

Abstract

Western blotting is a powerful tool for assessing the presence and modification of a protein. No suitable reference proteins for barley (Hordeum vulgare L.) western blotting has identified. In this study, we compared the quality of the total protein extracted from barley leaves, roots, and seeds using the Tris, QB, direct, phenol, TCA and modified TCA methods. The results confirmed the Tris, direct and modified TCA methods were optimal for seeds, young tissues and mature tissues protein extracting, respectively. The stability and sensitivity among the three reference proteins, i.e., plant actin, heat shock protein 90, and histone H3, during barley western blotting among were compared in different tissues/organs at various stages of development and/or stress. The results showed that plant actin was the most sensitive and the most constantly expressed among all the reference proteins tested in all barley organs. A linear relationship (R=0.9710) of the plant actin signal intensity with the sample amount was detected when the total protein ranged within 10-160 µg. The optimized barley western blotting procedure was used to investigate the response of H3K27me1 and H3K27me3 to waterlogging in barley. Under waterlogging, the expression of H3K27me1 was repressed in the stems but promoted in the roots, while the expression of H3K27me3 was repressed in both the leaf and stem. This is the first report about H3K27 methylation modifications displayed the organspecific response patterns to waterlogging in barley.

Comparative transcriptomic and epigenetic analyses reveal conserved and divergent regulatory pathways in barley response to temperature stresses

“…After centrifugation at 12,000 g for 15 min, the supernatant was collected for western blot analysis. Western blot detection was performed according to Wang et al (2018). Primary antibodies, including anti‐H3K9ac (#06‐942, Millipore), anti‐H3K4me3 (#07‐473, Millipore,), and anti‐H3 (#PL030349R, Pllabs), as well as the secondary antibody anti‐rabbit IgG (#AP132F, Millipore), were used for immunoblotting.…”