ABSTRACT
Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/drug effects , Brassinosteroids/pharmacology , Gene Expression Regulation, Plant , Nuclear Proteins/metabolism , Plant Growth Regulators/pharmacology , Signal Transduction , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Chromatography, Affinity , DNA-Binding Proteins , Mass Spectrometry , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Molecular Sequence Annotation , Nuclear Proteins/genetics , Phosphorylation , Protein Binding , Protein Interaction Mapping , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Transport , ProteolysisABSTRACT
Both zhenbao pills II and III are Mongolian medicine of a kind. Zhenbao pill II was the artificial syntheses and zhenbao pill III was made from natural materials. In this paper, the flavonoids of Mongolian medicine zhenbao pills II and III were extracted by Soxhlet apparatus with methyl alcohol as extracting agent, the colorimetric method was applied to the determination of flavonoids, and the experimental procedure was studied with zhenbao pill II as the test sample. The result showed that the linear range of quantitative determination was 8.05-48.28 microg x mg(-1). The standard addition recovery (SAR) was 99.49%-100.50%. The RSD (n = 3) was 0.54%. The range of contents of flavonoids was 1.47-1.55 mg x g(-1) in zhenbao pill II and was 2.88-3.00 mg x g(-1) in zhenbao pill III. This method was simple and accurate with good reproducibility, and is suitable for the determination of flavonoids in all kinds of pills. The contents of flavonoids can be used to prove whether zhenbao pill is artificial syntheses or natural material.
