Purification And Immunohistochemical Localization Of Water Stress Induced Proteins In Intercellular Fluids Of Wheat Leaves

Apoplast, as an important compartment outside of plasma membrane, is the connector between the environment and cells and is the source of various primary signals. Apoplast plays a key role in minerals transport, signal transduction, hormone distribution and the interaction between pathogens and the host. It is the decisive factor in the regulation of cell differentiation, organ genesis, and growth development of plants. The molecular events in intercellular washing fluids (IWF) are important for studying physiological and biochemical responses of plants to adverse stresses. In this study, Wheat xiaoyan6 was used as sample to study the protein change in wheat IWF induced by water stress. Those seedlings were treated with 27.5% PEG-6000 solution in advance and IWF was extracted by vacuum infiltrated with distilled water.The proteins of IWF after treatment with 27.5% PEG were extracted and then separated by SDS-PAGE, the results showed that three subunits with molecular weight 50kD, 36kD and 21kD were induced after treatment with 27.5%PEG for 4, 6 and 8 days and disappeared after rewatering 2 days. Thus, in conclusion, three water stress induced proteins had a correlation with the response of wheat to drought stress.The 50kD protein was purified by electro elution protocol and was analyzed by MALDI-TOF-MS and LC-MS. The obtained peptide mass fingerp rinting (PMF) was aligned in the protein data bank, indicating that it was the degradation products from the large subunit of Rubisco. Therefore, the further identification should be done in the following.The IWF was analyzed by Western-blotting with rabbit-anti-Rubisco antibody. The result indicated that the large subunit of Rubisco was detected after treatment with 27.5% PEG at 4, 6, and 8d. However, the 50kDa fragment could not be found at 2, 4, 6 and 8 days after treatment with water and 2 days after rewatering. In this study, the immunohistochemical localization of Rubisco LSU was carried out by using the rabbit-anti-Rubisco antibody of the IWF after treatment with 27.5% PEG. And plenty of degradation products of large subunit of Rubisco were found in the IWF by water stress, but nothing in the control.In order to study the degradation way of the Rubisco LUS by drought stress in plants, the subcellular immunogold localization should be done for the further study.