"BACKGROUND: Proteomics is a high-throughput approach that could provide comprehensive understanding of complex developmental processes. Holm oak (Quercus ilex L.) is a dominant tree in Mediterranean natural communities. Oak seeds are recalcitrant. Seed proteome and germination under stress are studied predominantly in cereals and model plants, but there is very limited information on forest species.OBJECTIVES: This project aims to study germination and early seedling growth of Holm oak at the proteome level, focusing particularly on how these processes are affected by drought. Emphasis will be placed on phosphorylation and ubiquitination as major posttranslational modifications (PTM). Proteome analysis will be complemented with data at the metabolite and transcript levels and with cytochemical observations.METHODOLOGY: Holistic and targeted approaches will be combined.Holistic approach – by using gel and gel-free, including label free quantitation approaches coupled to MS, changes in the protein and PTM profiles associated with the germination and stress responses will be analyzed. PTM will be detected by specific staining or Western blot. Appropriate statistical analysis, algorithms, databases and repositories will be used.Targeted approach – the oxidative status of the tissues will be monitored and more detailed analysis of some proteins of interest will be performed at the transcript level. Analyses at the metabolite level will include carbohydrates (ethanol extraction, GC-MS), aminoacid derivates (ethanol extraction, HPLC quantification), some secondary metabolites (GC or HPLC). Histochemical staining of carbohydrates, phenolic compounds, and H2O2 will also be performed.EXPECTED RESULTS: To gain deeper knowledge of the response to drought in a recalcitrant seed- producing tree species at the earliest developmental stages. The relevance of specific PTMs will be evaluated. Proteomic data will be validated by complementary targeted studies."