Analysis of the desiccation phosphoproteome in the resurrection plant Craterostigma plantagineum.

• Applicants: Dr. Horst Röhrig; Dr. Jürgen Schmidt
• Subject Area: Plant Cell and Developmental Biology ; Plant Physiology
• Term: from 2006 to 2012
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 34097530

Reversible protein phosphorylation/dephosphorylation is crucial for regulation of many cellular events and increasing evidence indicates that this post-translational modification is also involved in the process of acquisition of desiccation tolerance. In the current research program an approach was developed for the analysis of leaf phosphoproteins of the desiccation tolerant resurrection plant Craterostigma plantagineum including a phosphoprotein enrichment step by metal oxide affinity chromatography (MOAC), 2-D PAGE, staining with a phosphoprotein-specific dye and subsequent protein identification via MS. Using this strategy several potential phosphoproteins were identified. This continuation proposal is based in part upon these established tools and aims to a comprehensive analysis of the desiccation phosphoproteome in callus tissue of C. plantagineum. The callus is more advantageous than using leaf tissue due to increased detection sensitivity for non-chloroplastic proteins, the possibility to perform the comparative analyses between desiccation tolerant and desiccation sensitive tissues but also to analyse more precisely early and late events during dehydration. In addition to methods basing on gel-electrophoresis and subsequent MALDI-TOF/TOF MS we will apply also gel-free approaches including protocols for the enrichment of phosphopeptides and subsequent nanoLC combined with an ion trap MS for the detection of phosphorylation sites by neutral loss analysis.In our recent joined project, we analysed the expression and post-translational modification of CDeT11-24, a LEA-like protein which is correlated with desiccation in C. plantagineum. Our results show a biphasic expression of the protein. The plant hormone ABA which is responsible for the early plant reactions to water stress induces expression of CDeT11-24 but, phosphorylation of the protein occurs late during the desiccation process. Using “in-gel” kinase assays we could identify CDeT11-24 phosphorylating kinase activities which correlate with desiccation. Therefore, in a further part of this proposal we want to purify the corresponding enzymes and identify the proteins by MS. Together with the analyses of structural transitions and protein-protein interactions induced by CDeT11-24 phosphorylation these in vitro studies will be a significant contribution to demonstrate the importance of protein phosphorylation for the process of desiccation tolerance.

• DFG Programme: Research Grants
• Participating Person: Professorin Dr. Dorothea Bartels