Characterization of brassinosteroid-regulated proteins in a nuclear-enriched fraction of Arabidopsis suspension-cultured cells

Abstract

To identify nuclear proteins involved in the brassinosteroid (BR) signaling pathway, a targeted proteomic approach was applied to Arabidopsis thaliana suspension-cultured T87 cells. Cell growth was promoted by 0.1 μM brassinolide (BL) and inhibited by 5 μM brassinazole (Brz). Analysis of BR-regulated proteins in nuclear-enriched fractions was carried out using two-dimensional polyacrylamide gel electrophoresis with a special fluorescent dye. Proteins of interest were identified by correlating normalized spot volume of proteins on the gels with cellular BR level (Brz-treated cells, extremely low level of BRs; control cells, normal level of BRs; BL-treated cells, high level of BRs). A number of BR-responsive proteins were detected and some of these proteins were identified by nano-liquid chromatography-tandem mass spectrometry after enzymatic digestion. Fluctuations in eight identified nuclear proteins in BL-treated cells were investigated in the first 12 h of treatment. Three nuclear BR-responsive proteins, Nucleosome Assembly Protein (NAP) 1;1, Band 7 Family Protein, and Vernalization Independence 3, significantly decreased during this time. Meanwhile, NAP1;2, S-Adenosylmethionine Synthetase 2, and 60S Ribosomal Protein L14 increased markedly over time. Since some of these proteins are reportedly related to chromosome remodeling, cell growth induced by BL may involve chromatin remodeling. Interestingly, NAP1;2 was found to be post-translationally modified in response to cellular BR levels. Our study of quantitative protein changes in the nucleus provides valuable insight into BR-induced cellular and physiological responses.