Plant secondary cell wall proteome analysis with an inducible system for xylem vessel cell differentiation

Plant cell walls are typically composed of polysaccharide polymers and cell wall proteins (CWPs). CWPs account for approximately 10% of the plant cell wall structure and perform a wide range of functions. Previous studies have identified approximately 1000 CWPs in the model plant Arabidopsis thaliana; however, the analyses mainly targeted primary cell walls, which are generated at cell division. In contrast, little is known about CWPs in secondary cell walls (SCWs), which are rigid and contain the phenolic polymer lignin. Here, we performed a cell wall proteome analysis to obtain novel insights into CWPs in SCWs. To this end, we tested multiple methods for cell wall extraction with cultured Arabidopsis cells carrying the VND7-VP16-GR system, with which cells can be transdifferentiated into xylem-vessel-like cells with lignified SCWs by dexamethasone treatment. We then subjected the protein samples to in-gel trypsin digestion followed by LC-MS/MS analysis. The different extraction methods resulted in the detection of different cell wall fraction proteins (CWFPs). In particular, centrifugation conditions had a strong impact on the extracted CWFP species, resulting in the increased number of identified CWFPs. We successfully identified 896 proteins as CWFPs in total, including proteases, expansins, purple phosphatase, well-known lignin-related enzymes (laccase and peroxidase), and 683 of 896 proteins were newly identified CWFPs. These results demonstrate the usefulness of our CWP analysis method. Further analyses of SCW-related CWPs could be expected to produce information useful for understanding the roles of CWPs in plant cell functions.