Functional phosphoproteomic profiling of phosphorylation sites in membrane fractions of salt-stressed Arabidopsis thaliana

Jue-Liang Hsu,Fong-Ku Shi,Ing-Feng Chang,Shu-Ying Wang,Lan-Yu Wang,Ching-Huang Lin,Kuo-Chieh Ho

doi:10.1186/1477-5956-7-42

Abstract

BackgroundUnder conditions of salt stress, plants respond by initiating phosphorylation cascades. Many key phosphorylation events occur at the membrane. However, to date only limited sites have been identified that are phosphorylated in response to salt stress in plants.ResultsMembrane fractions from three-day and 200 mM salt-treated Arabidopsis suspension plants were isolated, followed by protease shaving and enrichment using Zirconium ion-charged magnetic beads, and tandem mass spectrometry analyses. From this isolation, 18 phosphorylation sites from 15 Arabidopsis proteins were identified. A unique phosphorylation site in 14-3-3-interacting protein AHA1 was predominately identified in 200 mM salt-treated plants. We also identified some phosphorylation sites in aquaporins. A doubly phosphorylated peptide of PIP2;1 as well as a phosphopeptide containing a single phosphorylation site (Ser-283) and a phosphopeptide containing another site (Ser-286) of aquaporin PIP2;4 were identified respectively. These two sites appeared to be novel of which were not reported before. In addition, quantitative analyses of protein phosphorylation with either label-free or stable-isotope labeling were also employed in this study. The results indicated that level of phosphopeptides on five membrane proteins such as AHA1, STP1, Patellin-2, probable inactive receptor kinase (At3g02880), and probable purine permease 18 showed at least two-fold increase in comparison to control in response to 200 mM salt-stress.ConclusionIn this study, we successfully identified novel salt stress-responsive protein phosphorylation sites from membrane isolates of abiotic-stressed plants by membrane shaving followed by Zr4+-IMAC enrichment. The identified phosphorylation sites can be important in the salt stress response in plants.

Highlights

Under conditions of salt stress, plants respond by initiating phosphorylation cascades
Mapping of phosphorylation sites from membrane fractions of Arabidopsis through membrane shaving and Zr4+-IMAC bead enrichment In order to identify phosphorylation sites in a model organism, Arabidopsis thaliana was chosen as a source of plant material
Solubilization of membrane proteins using surfactants followed by enzymatic or chemical fragmentation prior to LC-Mass spectrometry (MS)/MS analysis is a common approach in the identification of membrane proteins

Summary

Introduction

Under conditions of salt stress, plants respond by initiating phosphorylation cascades. To date only limited sites have been identified that are phosphorylated in response to salt stress in plants. Under conditions of salt stress, plants respond by activating phosphorylation cascades. Mass spectrometry-based proteomic studies have identified salt-induced proteins in plants [7,8,9,10,11]. Both tools provide efficient ways to identify genes or proteins responsive to salt stress. The unique protein phosphorylation sites required for the plant response to salt stress have not been well characterized

Methods

Results

Conclusion