Differential proteomic analysis of soluble extracellular proteins reveals the cysteine protease and cystatin involved in suspension-cultured cell proliferation in rice

Extracellular matrix proteins play crucial roles in plant development, morphogenesis, cell division, and proliferation. To identify extracellular proteins that regulate cell growth, the soluble proteins of extracellular matrix were extracted from suspension-cultured rice cells for different lengths of time. The extract obtained from 3-d cultures was found to increase cells' fresh weight, while extracts from 6-d and 9-d cultures showed no effect on cells' growth. A comparative proteomic analysis was used to identify soluble extracellular proteins differentially expressed between 3 and 6 days in suspension-cultured cells. Ten unique protein spots were isolated and identified by mass spectrometry. Among them, included a cysteine protease (OsCP) and a putative cysteine protease inhibitor (cystatin, OC-I). OsCP has been down regulated in vivo using RNAi transgenic lines. The fresh and dry weight growth rates of OsCP RNAi cell lines were lower than empty vector control. Recombinant protein of OC-I could inhibit the OsCP protease activity, also it could inhibit the weight increase of suspension-culture cell as well as extracellular protease activity. These results suggest that OsCP and OC-I may be involved in the process of suspension-cultured rice cells proliferation.