Analysis of the desiccation phosphoproteome in the resurrection plant Craterostigma plantagineum.
Pflanzenphysiologie
Zusammenfassung der Projektergebnisse
To identify desiccation-related phosphoproteins a proteomic approach based on phosphoprotein enrichment, 2D SDS–PAGE and MALDI-TOF/TOF MS was applied using extracts from callus tissue of the resurrection plant C. plantagineum. Treatment of callus with ABA induces the expression of a set of genes comparable with those activated upon drying in the whole plant turning callus tissue from desiccation-sensitive to a tolerant stage. In order to dissect ABA signaling and drought stress, the callus was dried with or without ABA treatment and analyzed for changes in the phosphoproteome. Using this strategy we identified 99 different putative phosphoproteins, which were mostly not regulated by the different treatments. However, some phosphoproteins, regulated upon these treatments, represent so far not described proteins involved in the mechanisms of desiccation tolerance. One of the major desiccation-related phosphoproteins of C. plantagineum is the LEA-like protein CDet11-24. We show that CDeT11-24 is mostly disordered and that the protein is able to protect the two enzymes lactate dehydrogenase and citrate synthase against the damaging effect of desiccation. Furthermore, Escherichia coli cells overexpressing CDeT11-24 showed an increased viability after drying, showing a protective function of the protein also in whole cells. Surprisingly, lipid binding assays revealed that CDeT11-24 is able to interact with phosphatidic acid, although electrostatic repulsion could be expected due to the negative charge of the protein under the applied physiological conditions. Analysis with the CDeT11- 24 protein carrying a deletion within the N-terminal lysine rich sequence, the so-called K-segment, identified this region to be responsible for this interaction. Based on these findings, it is reasonable to assume, that the CDeT11-24 phosphoprotein has a physiological function in the context with desiccation tolerance. We could also establish an experimental setup for the identification of CDeT11-24 kinases in C. plantagineum extracts. Using this approach we were able to identify two different kinases showing homology to the alpha subunit of CK2 (CK2α) and the mitogen-activated triple kinase-like protein kinase (VIK), respectively. Since phosphorylation of CDeT11-24 is triggered independently of the protein induction by ABA and occurs during late stages of desiccation, both enzymes may be involved in the tolerance mechanisms during severe water stress. However, more experiments are required to provide further evidence for this hypothesis and to gain deeper insight into the functional involvement of these kinases.
Projektbezogene Publikationen (Auswahl)
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2010. Phosphoproteomic analysis of Craterostigma plantagineum upon abscisic acid and desiccation stress. PhD thesis. University Bonn
Facchinelli, F.
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2011. Comparative analysis of LEA-like 11-24 gene expression and regulation in related plant species within the Linderniaceae that differ in desiccation tolerance. New Phytologist 190. 75-88
van den Dries N, Facchinelli F, Giarola V, Phillips JR, Bartels D
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Modified metal-oxide affinity enrichment combined with 2D-PAGE and analysis of phosphoproteomes. In: Methods in Molecular Biology. Plant Kinases. Dissmeyer N, Schnittger A, eds (2011) Vol. 779. Humana Press. 273-286
Colby T, Röhrig H, Harzen A, Schmidt J
