Phosphoproteomic Analysis of <em>Craterostigma plantagineum </em>upon Abscisic Acid and Desiccation Stress

Abstract

Studies aimed at understanding the molecular basis of the desiccation tolerance in angiosperms have mainly focused on the South African plant Craterostigma plantagineum. C. plantagineum belongs to the Linderniaceae family, which comprises both desiccation tolerant and sensitive species. A recent study demonstrated the monophyly of the lineage that includes the genera Craterostigma and Lindernia. <br /> The ability of two Lindernia species to withstand severe water stress was investigated at the cellular level, leading to the conclusion that the close relatives Lindernia brevidens and Lindernia subracemosa display different phenotypes regarding the ability to survive desiccation: L. brevidens is desiccation tolerant, whereas L. subracemosa is not.<br /> The fact that a genus close to the model plant C. plantagineum displays such opposite phenotypes regarding the ability to survive desiccation provides a useful tool for deciphering the complex trait of the desiccation tolerance. This variability was exploited to analyse a candidate protein whose homologue CDeT11-24 of C. plantagineum has been implicated in the desiccation response and is considered to be related to the late embryogenesis abundant (LEA) proteins. The protein sequences of the Lindernia brevidens (Lb11–24) and Lindernia subracemosa (Ls11–24) counterparts were isolated. Sequence analysis identified conserved motifs common to other proteins known as stress responsive. <br /> The LEA-like protein CDeT11-24 was reported as one of the major phosphoproteins accumulating upon desiccation in the vegetative tissues of C. plantagineum. The phosphorylation status of the 11-24 proteins was dissected in response to the tissue priming by the plant hormone ABA and by desiccation treatment, revealing that ABA is able to induce the protein synthesis and that desiccation is necessary and sufficient to trigger its phosphorylation. However, the 11-24 homologue of the desiccation sensitive L. subracemosa is not phosphorylated as strongly as the desiccation-tolerant plants. The 11-24 protein is therefore regulated by phosphorylation and its phosphorylation correlates with the ability of the plants to withstand desiccation. The identification of the phosphorylation sites of the three homologues could then provide additional information about the distribution and conservation of the phosphorylatable residues, since they occur in proximity of predicted coiled-coil regions. The particular regulation and distribution of the phosphorylation led to the investigation of the potential interaction partners of the CDeT11-24 protein. An affinity chromatography-based approach could reveal that the CDeT11-24 protein interacts with itself in its unphosphorylated form, providing evidence for a phosphorylation-driven regulation of its oligomerisation.<br /> Finally, a more extensive screening was performed to identify protein whose phosphorylation is regulated in response to the ABA and desiccation treatment. In this study an approach based on phosphoprotein enrichment and 2D SDS–PAGE was applied on C. plantagineum callus tissue. Treatment of callus with ABA induces the expression of a set of genes comparable with that activated upon drying in the whole plant. The callus was dried with or without prior ABA treatment, in order to dissect the different contribution of ABA induction and drought stress on phosphoprotein changes. Moreover, the callus tissue presents the advantage of lacking in the photosynthesis-related proteins, which turned to be the main phosphoproteins identified in leaves, with the RuBisCO being the most abundant. This approach provided a list of candidate proteins whose phosphorylation is regulated during the treatments imposed and furnished novel elements involved in the mechanisms of the desiccation tolerance.