Comparative Proteomics Analysis of the Root Apoplasts of Rice Seedlings in Response to Hydrogen Peroxide

Background: Plant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H2O2) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H2O2-responsive proteins in the apoplast of rice seedling roots. Methodology/Principal Findings: 14-day-old rice seedlings were treated with low concentrations (300 and 600 mM) of H2O2 for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H2O2 were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H2O2 conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H2O2-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H2O2 stress. Conclusions/Significance: The present study is the first apoplast proteome investigation of plant seedlings in response to H2O2 and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H2O2 response.