Features of protein isolation for pea Pisum sativum L. root proteome analysis during symbiosis with rhizobia

Pea Pisum sativum L. is a convenient model to study the molecular-genetic mechanisms of nitrogen-fixing symbiosis establishment with rhizobia, because a representative collection of mutants, blocked at different stages of symbiosis development was obtained. A comparative analysis of the proteomes of the wild type cultivars and lines of peas and mutants can be a useful approach for carrying out studies aimed on at identification and further analysis of regulators controlling the formation of nitrogen-fixing nodules. However as the review of modern literary data shows, studies of differential proteome changes in pea roots during symbiosis are almost not performed. Sample preparation is a key stage in proteomic studies. The quality of gels obtained after 2-D electrophoresis and the opportunity of following analysis depend on protein isolation efficiency from the tissues and purification from accompanying substances. Our work is aimed on finding the most effective method of protein isolation from Pisum sativum roots inoculated with rhizobia, which might be applied for carrying the 2-D electrophoresis. Special requirements aimed at separation stages minimization important for protein stability, as well as the efficient removal of contaminants which can negatively affect the quality of separation and the subsequent evaluation of qualitative and quantitative changes in the protein synthesis are necessary for proteomics. Analysis of data revealed a number of possible methods for the protein isolation from plant tissues. A comparison of three methods of the proteins isolation using the commercial protocol from Bio-Rad; the method based on treatment with phenol and ammonium acetate as well as the trichloroacetic acid application. Pea plants of cv. Frisson were used in our work, the strain Rhizobium leguminosarum bv. viciae CIAM1026 was used for inoculation. After protein isolation from the wild-type cv. Frisson roots of pea seedlings inoculated with rhizobia (1 day after inoculation) using three methods and consequent 2-D electrophoresis, it was shown that the best results are achieved using the method with phenol following by ammonium acetate precipitation. The gels were analyzed for trace presence that made it difficult to search for different proteins, the efficiency of total protein isolation and possible degradation products. Using this selected method, the differential 2-D electrophoresis of extracted proteins was carried out with fluorescent Cy2 and Cy5 labels based on isoelectric focusing of proteins using strips with a pH range of 3-10 and subsequent separation in a polyacrylamide (PAGE) gel. The analysis showed that when proteins were isolated using phenol and ammonium acetate, it was possible to obtain rather representative proteomes of the roots of pea seedlings. The differential 2-D electrophoresis allowed to see the differences between the control samples (non-inoculated roots) and the samples inoculated with rhizobia (inoculated roots). This method may be recommended for further proteomic studies in pea roots.