Analysis Of Extracellular Proteins Of Rice Suspension-Cultured Cells

Plant apoplast is a very important structure and apoplast proteins play crucial roles in plant development, morphogenesis, cell division and proliferation. It has reported that there were some conditional factors in suspension-cultured cell conditional medium that could promote cell division. In this paper, a comparative proteomic analysis was used to find more proteins that regulate cell growth from the medium of rice suspension-cultured cell.The rice (Oryza sativa L) suspension-cultured cell system was established. According to the cell growth curve, the proteins of extracellular matrix were extracted from suspension-cultured rice cells for different lengths of time. To evaluate the level of cytoplasmic contamination, two different approaches were used. First, similarly to most studies of extracellular proteins, malate dehydrogenase (MDH) activity was assayed as a quantitative marker of intracellular contamination. Second, we examined extracts for the presence of tubulin, which is a major intracellular protein detectable by SDS-PAGE and immunoblot using a monoclonal antibody. Together, these data indicate that the extracellular extracts used in the proteomics analysis were free of cytoplasmic contamination. The activity of the extracts obtained from different culturing periods was tested. The extract obtained from 3-day-old cultures medium was found to increase cells fresh weight, while extracts from 6- and 9-day-old cultures medium showed no effect on cells growth.A comparative proteomic analysis was used to identify extracellular matrix proteins differentially expressed between 3 and 6 days in suspension-cultured rice cells. Eleven protein spots were isolated and ten spots were identified by mass spectrometry, in fact, they belong to seven proteins. All spots have an N-terminal signal peptide and no transmembrane domains when we predicted by bioinformatics analysis. This consists with the idea that these proteins are secreted into the extracellular medium.The genes for the cysteine proteases (OsCP), cystatin (OsCPI) and plant basic secretory protein (OsBSP) were transiently expressed as fusions with yellow fluorescent protein in onion cells to study their subcellular localization. The results indicate that both OsCPI and OsBSP are detected in the cell wall area, and OsCP is detected with vesicle. The real-time PCR were used to detecte the OsCP, OsCPI and OsBSP genes expression at different cultured time, and the results indicated that OsCPI and OsBSP were changed in RNA that consistent with the protein level, but no the OsCP. This difference may be caused by the translation modification. We also used SDS-PAGE and LC/MS-MS to analysis the components of 3-day extracellular matrix protein, and got some well-known extracellular proteins, such asα-amylase, peroxidase et al.Among the changed proteins, the OsCP was increased in abundance at 3 days extracellular proteins which could promote the cell growth, and OsCPI was only detected in the 6-day extract. This interesting phenomenon takes our attention, and the functions of OsCP and OsCPI were analyzed. We gained three OsCP RNAi suspension-cultured cell lines by transgenic. The results of real-time PCR indicated that the the OsCP gene expression were descreased in all of OsCP RNAi cell lines, and the three lines were indepent lines by sourthern-blot analysis. The increase rate of fresh and dry mass of OsCP RNAi suspension-cultured cell was descreased. The cell diameters of them were not different with the empty vector. At the same time, we purified the OsCPI fusion protein and the inhibition activity showed that the purified OsCPI fusion protein could inhibite the papain activity. When 10-7 and 10-8mol/L OsCPI proteins were added to the cultured medium, the increase rate of fresh and dry mass of suspension-cultured cell was descreased, the GST protein as a control. 10-7 mol/L OsCPI protein has no effect on tobacco BY-2 cell lines and this indicated that the cell growth inhibition of OsCPI proteins was specific.In conclusion, a proteomic analysis was used to find the proteins that may regulate cell growth from the extracellular matrix of rice suspension-cultured cell, OsCP and OsCPI were used for functional analiysis. The results indicated that as the extracellular proteins, the proteases and inhibitor were predicted to modulate cell growth and proliferation.