Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

Abstract

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel-based and a LC MS/MS-based proteomics method. Two-day-old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two-phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel-based proteomics, four and eight protein spots were identified as up- and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up- and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low-abundance proteins could be identified by the LC MS/MS-based method. Three homologues of plasma membrane H(+)-ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H(+)-ATPase protein.

Endoplasmic Reticulum Stress Response

“…As ER-resident molecular chaperones, both calreticulin and calnexin from soybean are induced by ER stressors, such as tunicamycin and AZC (Irsigler et al, 2007). Under hyperosmotic conditions, soybean calnexin is up-regulated and accumulates in the plasma membrane (Nouri & Komatsu, 2010).…”

Proteomics Approach for Identifying Abiotic Stress Responsive Proteins in Soybean

“…The gel-free technique has the advantage of being capable of identifying low-abundance proteins, proteins with extreme molecular weights or pI values, and hydrophobic proteins that cannot be identified by using gel-based technique. A combination of gel-based and gel-free proteomics has been used for identification of soybean plasma membrane proteins under flooding (Komatsu et al, 2009a;Nanjo et al, 2010) or osmotic stress (Nouri & Komatsu, 2010), suggesting that these two methods are complementary to one another for protein identification. Methods for protein identification are not usually organism specific, and they can be applied to a wide range of living organisms in addition to soybean.…”

“…The increased abundance of SOD and thioredoxin in soybean plants correlates with enhanced tolerance of the plants to osmotic stress. Nouri & Komatsu (2010) investigated the soybean plasma membrane proteome under osmotic stress by using gel-based and gel-free proteomics approaches. They adapted a twophase partitioning method to purify plasma membranes from soybean seedlings that had been treated for two days with 10% PEG.…”

“…In all such experiments, the purification of protein extracts and the verification of their purity determine the validity of the results of the proteomics analysis. Plasma membrane extracts were purified by using a two-phase partitioning method and their purity was verified by measurement of the activity of P-type ATPase (Komatsu et al, 2009a;Nouri & Komatsu, 2010). A cell wall fraction was obtained by using calcium chloride, and its purity was confirmed by assaying the activity of glucose-6-phosphate dehydrogenase .…”

“…The extraction methodologies explained here are generally used in separation of total proteins by classical proteomics approaches such as 2-DE. In the case of organelle proteomics particularly that of membrane proteomics, a different extraction procedure is required that involves modifications to dissolve hydrophobic proteins and additional purification steps (Komatsu et al, 2009a;Nouri & Komatsu, 2010). Furthermore, when studying protein-protein interactions, it is necessary to extract protein complexes by using buffers with less or no detergent to get the proteins in their native states.…”