The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes. How BR signaling regulates diverse processes remains unclear. To understand the molecular details of BR responses, we performed a proteomics study of BR-regulated proteins in Arabidopsis using two-dimensional DIGE coupled with LC-MS/MS. We identified 42 BR-regulated proteins, which are predicted to play potential roles in BR regulation of specific cellular processes, such as signaling, cytoskeleton rearrangement, vesicle trafficking, and biosynthesis of hormones and vitamins. Analyses of the BR-insensitive mutant bri1-116 and BR-hypersensitive mutant bzr1-1D identified five proteins (PATL1, PATL2, THI1, AtMDAR3, and NADP-ME2) affected both by BR treatment and in the mutants, suggesting their importance in BR action. Selected proteins were further studied using insertion knock-out mutants or immunoblotting. Interestingly about 80% of the BR-responsive proteins were not identified in previous microarray studies, and direct comparison between protein and RNA changes in BR mutants revealed a very weak correlation. RT-PCR analysis of selected genes revealed genespecific kinetic relationships between RNA and protein responses. Furthermore BR-regulated posttranslational modification of BiP2 protein was detected as spot shifts in two-dimensional DIGE. This study provides novel insights into the molecular networks that link BR signaling to specific cellular and physiological responses.

Signal transduction involves posttranslational modifications and protein-protein interactions, which can be studied by proteomics. In Arabidopsis, the steroid hormone (brassinosteroid (BR)) binds to the extracellular domain of a receptor kinase (BRI1) to initiate a phosphorylation/ dephosphorylation cascade that controls gene expression and plant growth. Here we detected early BR signaling events and identified early response proteins using prefractionation and two-dimensional (2-D) DIGE. Proteomic changes induced rapidly by BR treatments were detected in phosphoprotein and plasma membrane (PM) fractions by 2-D DIGE but not in total protein extracts. LC-MS/MS analysis of gel spots identified 19 BR-regulated PM proteins and six proteins from phosphoprotein fractions. These include the BAK1 receptor kinase and BZR1 transcription factor of the BR signaling pathway. Both proteins showed spot shifts consistent with BR-regulated phosphorylation. In addition, in vivo phosphorylation sites were identified for BZR1, two tetratricopeptide repeat proteins, and a phosphoenolpyruvate carboxykinase (PCK1). Overexpression of a novel BR-induced PM protein (DREPP) partially suppressed the phenotypes of a BR-deficient mutant, demonstrating its important function in BR responses. Our study demonstrates that prefractionation coupled with 2-D DIGE is a powerful approach for studying signal transduction. Molecular & Cellular Proteomics 7:728 -738, 2008.Perception and response to extracellular signals are crucial for growth and survival of all organisms. Signals are transduced intracellularly through mechanisms that include posttranslational protein modification and protein-protein interaction. Detection of such signaling events using proteomics methods can identify proteins involved in signal transduction but is technically challenging because of the low abundance of signaling proteins. Brassinosteroids (BRs) 1 are plant hormones that play important roles in multiple plant developmental processes. Mutant plants with a defect in BR synthesis or signal transduction show a wide range of phenotypes including dwarfism, reduced fertility, light-grown morphology in the dark, and delayed senescence (1). BRs structurally resemble animal steroid hormones but act through a distinct signaling mechanism (2). Although animal steroid hormones are perceived by nuclear receptors, BRs are perceived by a cell surface receptor-like kinase named BRI1 (2, 3). BRs bind to the extracellular domain of BRI1 to activate its kinase and downstream BR signal transduction (4), which involves another receptor-like kinase named BAK1 (5, 6); a GSK3-like kinase, BIN2 (7); a phosphatase, BSU1 (8); and two transcription factors, BZR1 and BZR2 (also known as BES1) (9, 10). In the absence of BR, the inhibitory BIN2 kinase phosphorylates BZR1 and BZR2, and phosphorylation inhibits the function of BZR1 and BZR2 through multiple mechanisms, which include inhibition of DNA binding activity, degradation by the proteasome, and cytoplasmic retention by the 14-3-3 proteins that b...

The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B-tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B-sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors. Mass spectrometic analysis of posttranslational modifications on histone proteins demonstrates that UV-B-tolerant lines exhibit greater acetylation on N-terminal tails of histones H3 and H4 after irradiation. These acetylated histones are enriched in the promoter and transcribed regions of the two UV-B-upregulated genes examined; radiation-sensitive lines lack this enrichment. DNase I and micrococcal nuclease hypersensitivity assays indicate that chromatin adopts looser structures around the selected genes in the UV-B-tolerant samples. Chromatin immunoprecipitation experiments identified additional chromatin factor changes associated with the nfc102 test gene after UV-B treatment in radiation-tolerant lines. Chromatin remodeling is thus shown to be a key process in acclimation to UV-B, and lines deficient in this process are more sensitive to UV-B.