Conclusions and Outlook

The results presented here show that plant species belonging to the Linderniaceae family, a clade containing different desiccation tolerant angiosperms, display different phenotypes regarding the ability to tolerate desiccation. Lindernia brevidens is desiccation-tolerant whereasLindernia subracemosa is desiccation-sensitive.

This intra-genus variability can be exploited as a tool for studying the complex trait of the acquisition of desiccation tolerance in plants. Focusing on other candidate proteins predicted to have a role in the desiccation response could reveal mechanisms that are exclusive to the desiccation tolerant species.

The sequences of the ABA- and desiccation-inducible proteins Lb11–24 and Ls11–24 ho-mologue to theCraterostigma plantagineum CDeT11–24 protein were isolated. Secondary structure predictions revealed the the 11–24 proteins are natively unfolded and the disorder is mostly confined to the N-terminal part. Sequence analysis identified conserved motifs

common to other proteins known as stress responsive, even if the conservation among the sequences is poor. Interestingly the sequence recognized by the alignment search is confined in the more conserved C-terminal part, indicating that this domain is possibly relevant for the function of the protein.

The advantage of comparing close relatives by looking at candidate proteins is more evident when looking at the phosphorylation status of the 11–24 proteins. It was shown that the protein is induced as a consequence of an early signal involving the ABA response and phosphorylated upon desiccation. The picture coming out from this study reinforces the role of the 11–24 protein fromCraterostigmaand Lindernia that is linked to the response of these plants to severe water stress. The 11–24 protein is regulated by phosphorylation and its phosphorylation correlates with the ability of the plants to withstand desiccation, since L. subracemosa is not phosphorylated as strongly as the protein from desiccation-tolerant plants. The analysis of the phosphorylation status of the 11–24 proteins from the desiccation-tolerant tissues of the seeds would offer additional informations to verify this model.

The identification of the phosphorylation sites provided additional clues about the putative function of the phosphorylation event, since it occurs in proximity of predicted coiled-coil regions. Due to the ability of phosphorylation to influence coiled-coil interactions, the individuation of interaction partners modulated by phosphorylation would confer valuable informations in understanding the role of the CDeT11–24 protein. In this work could be demonstrated that CDeT11–24, in its unphosphorylated form, is able to oligomerize. Fu-ture studies should focus on the function this interaction can have and test the importance of the coiled-coil stretches. In addition, alternative approaches that do not restrict the search to proteinaceous interaction partners have to be adopted.

The MOAC-based phosphoprotein enrichment turned out to be a valuable tool for a low to middle throughput analysis of the phosphoproteome ofC. plantagineum, delivering a list of candidate proteins that can be the starting point of future research. The use of the callus system provided an advantage over green tissues for the identification of less abundant proteins that would otherwise be masked by the abundant photosynthesis-related proteins.

In order to validate the candidate proteins regulated by phosphorylation, antibodies should be produced to confirm the changes in their phosphorylation status by immunoprecipiation.

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, whereasL. subracemosa is not.

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 ofC. 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. The primary structure of the candidate protein 11–24 from theseLindernia species was analysed in terms of amino acidic sequence prop-erties. Sequence analysis identified conserved motifs common to other proteins known as stress responsive.

Secondary structure predictions revealed the the 11–24 proteins are natively unfolded and the disorder is mostly confined to the N-terminal part, whereas the C-terminal is more conserved among the homologues and contains the motif common to the stress-responsive proteins.

The LEA-like protein CDeT11–24 was reported as one of the major phosphoproteins accu-mulating upon desiccation in the vegetative tissues ofC. plantagineum. The phosphory-lation status of the 11–24 proteins was dissected in response to the tissue priming by the plant hormone ABA and by desiccation treatment, providing evidence of the advantage of comparing close relatives by looking at candidate proteins. It could be concluded 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. To do this an antibody was raised against the candidate protein. A biochemical approach using the bait protein as interactor immobilised on an affinity column was performed. The affinity chromatography could reveal that the CDeT11–24 protein interacts with itself in its unphosphorylated form, providing evidence for a phosphorylation-driven regulation of its oligomerisation.

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 onC. 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.