Toward characterizing germination and early growth in the non-orthodox forest tree species Quercus ilex through complementary gel and gel-free proteomic analysis of embryo and seedlings.

By using two complementary proteomics, gel-based and gel-free (shotgun) approaches, the protein profiles of the non-orthodox forest tree species Quercus ilex seeds during germination and early seedling growth have been compared. Proteins were extracted from embryo axis, radicle and shoot tissues at different developmental stages. Proteins were subjected to one- and two-dimensional gel electrophoresis. A multivariate analysis (PCA) revealed that SDS-PAGE clearly separated germination (0-24 h post-imbibition), postgermination (72-216 h post-imbibition) and early seedling growth stages (2 weeks post-imbibition). Image analysis of the two-dimensional gels revealed a total of 732 spots, 103 of which were significantly variable among developmental stages. After MALDI-TOF/TOF MS analysis, 90 spots were identified, belonging to six main functional categories: carbohydrate, amino acids, energy, and protein metabolism, biosynthesis of secondary metabolites, and redox processes. The gel-based approach disclosed important metabolic changes that occurred in the holm oak seed after the germination. However, few proteins were significantly altered during the germination period (from 0 h to 24 h post imbibition) and, because of that, a further shotgun analysis was therefore used to analyse changes in the protein profile during seed germination. Up to 1250 proteins could be confidently identified, with 153 being variable. They belonged to the main functional categories of carbohydrate, amino acids and secondary metabolism, protein degradation, and responses to abiotic stress. The accumulation of proteases and amino acids metabolism proteins in mature seeds can be reflecting the production of energy from the mobilization of storage proteins to start germination. These results, therefore, corroborate the hypothesis that the mature non-orthodox seeds of Q. ilex have all the machinery necessary for rapidly resuming metabolic activities and starting the germination process, in contrast to that occurs in orthodox seeds, which metabolic activity ceases in mature dry seeds. The use of a genus-specific database combined with the public Viridiplantae database improved the quality and quantity of protein identification in this orphan species. In addition, both proteomics approaches (gel-based and shotgun) were complementary, with shotgun increasing by over two-fold the coverage of the proteome analysed. Both approaches provided similar results and supported the same conclusions on the metabolic switch experienced by the seed upon germination. SIGNIFICANCE: The optimal seed germination is a prerequisite for successful seedling establishment and plant vigour, being of great relevance in the case of crops and commercial woody plants. By using a complementary gel-based and gel-free proteomic strategy we have study the protein profiles of the non-orthodox forest tree species Quercus ilex seeds during germination and early seedling growth. The contribution of this work is of great importance, due to the complemented proteomic approaches giving similar clues to the metabolic state of the mature Q. ilex seed before the germination starts, and the metabolic switch experienced by the imbibed acorn until the seedling is established.