Medicago marina (L.) seed: unravelling mechanisms controlling germination and dormancy

The general aim of this thesis is to understand the complex and unknown mechanisms underlying dormancy and germination of Medicago marina (L.), a Mediterranean species distributed along the whole coasts of Italian peninsula and adapted to living in a specialized life habitats, like the dunes. Seeds of this species show strong dormancy that prevents germination. Results obtained during the first period of PhD program are published in a paper by Scippa et al., (2011). An integrated approach of physiological analyses and proteomics was used to investigate the mechanisms that control M. marina dormancy/germination and that underlie stress tolerance. First, was evaluated the effects on dormancy breaking of the following treatments: mechanical scarification, freezing at -20 °C, storage for 4 months and heating at 100 °C for 1 h. Mechanical scarification and freezing were the most effective treatments in overcoming dormancy. The role of abscisic acid (ABA) in M. marina dormancy was studied by ELISA immuno-enzymatic assay, determining the ABA content of germinated and non germinated mature (control) and treated seeds. The level of ABA was higher in treated seeds than in control seeds; the most significant increase occurred in the heated seeds. A comparison of the ABA level in the germinated, control and treated seeds suggests that different mechanisms modulate ABA content in response to different stresses, and that a specific ABA-signaling pathway regulates germination. Proteomic analysis revealed 46 proteins differentially expressed between treated and untreated seeds; 14 of these proteins were subsequently identified by mass spectrometry. Several of the proteins identified are important factors in the stress response, and are involved in such diverse functions as lipid metabolism, protein folding and chromatin protection. Lastly, an analysis of the phosphoproteome maps showed that the function of many proteins in seeds subjected to temperature treatment is modulated through post-translational modifications. Taken together, the data obtained showed that M. marina seeds adopt a dormancy strategy based on a hard seed coats, known as hardseededness, which prevent water uptake and germination; also, dormancy it can be regulated by other complex mechanisms beside seed coats, such as thermoinhibition, a delaying mechanisms of germination adopted by different species during late summer or early autumn. Starting from these first results, the PhD project proceeded in further investigations of mechanisms involved in the control of germination timing and dormancy establishment, due to the mechanical constraints of the micropylar endosperm, besides the seed coat. In order to accomplish this aim, the weakening of the endosperm was analyzed trough the puncture force measurements in seeds subjected to mechanical scarification and ABA treatment, along a 22 hours time course of imbibition. In addition to that, the effect of ABA and gibberellic acid treatments on germination rate and speed, were analyzed by performing germination test. Results obtained from the puncture force and germination tests for scarified seeds treated or not with ABA, revealed that the endosperm weakening, occurring in M. marina seeds before the germination, is delayed by ABA, which deferred also the germination of treated seeds. Then, the using of 2D analysis coupled whit the multivariate statistical analysis allowed the individuation of 95 spots that represent specific markers of different physiological states and might be expressed in seeds when dormancy is overcome in order to complete germination. First results suggest that the micropylar endosperm can contribute to the establishment of M. marina physical dormancy, that once again, it can be removed by mechanical scarification. Moreover, abscisic acid seems to be strongly involved in regulation of germination timing, by its action on micropylar endosperm weakening.