Abstract
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 gene-specific 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.
Highlights
The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes
Activation of the receptor kinases initiates a signaling cascade that leads to dephosphorylation and activation of the transcription factors BZR1 and BZR2 [13, 14] likely through inhibiting the glycogen synthase kinase 3-like kinase BIN2 or activating the phosphatase BSU1 [15, 16]
Plant Materials and Growth Conditions—The BR-deficient mutant det2-1 [48], BR-insensitive mutant bri1-116 [4], transgenic plants expressing the mutant bzr1-1D gene fused to cyan fluorescence protein [49] were in the Arabidopsis thaliana Columbia ecotype background
Summary
Plant Materials and Growth Conditions—The BR-deficient mutant det2-1 [48], BR-insensitive mutant bri1-116 [4], transgenic plants expressing the mutant bzr1-1D gene fused to cyan fluorescence protein (mBZR1-CFP) [49] were in the Arabidopsis thaliana Columbia ecotype background. The supernatant was mixed with an equal volume of ice-cold phenol (Tris-buffered, pH 7.5–7.9) and centrifuged at 20,000 ϫ g for 15 min at 4 °C to separate phenol and aqueous phases. Postelectrophoretic staining with deep purple (GE Healthcare) and SYPRO Ruby (Bio-Rad) fluorescent dyes was performed essentially according to the manufacturer’s instructions, and subsequent scanning was performed using the same setting as that used for Cy3-labeled sample. Manual editing was performed in the biological variation analysis module to ensure that spots were correctly matched between different gels and were not contaminated with artifacts, such as streaks or dust. Specific protein spots were washed twice with 50% acetonitrile in 25 mM ammonium bicarbonate (NH4HCO3), vacuum-dried, rehydrated in 8 l of digestion buffer (10 ng lϪ1 trypsin in 25 mM NH4HCO3), and covered with the minimum volume of NH4HCO3. Three PCR replicates from different biological samples were performed
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