Exploring softwoods fiber formation process in Douglas-fir (Pseudotsuga menziesii) in-vitro model system using proteomics approach

Background: Preudotsuga menziesii (Douglas-fir) is one of the most economically important timber species in Pacific Northwest. This demand has put a pressure on the wood industry to develop more sustainable strategies to improve yields and wood quality. One possible way to address this goal is a genetic modification of Douglas-fir creating faster to grow, pathogen resistant and most importantly with improved wood quality tree line. To achieve such an aim is to first characterize all involved molecular pathways and future modification targets. This can be especially difficult in case of a non-modal organism such as Douglas-fir. Without organism genome sequenced, another approach used in this tape of studies in a direct investigation of involved proteins my means of mass spectrometry. Comparative analysis of proteome of wood fibers, at different developmental stages, can be used to get an insight into the mechanism behind wood formation.;Methods: In order to study wood fiber formation process in Douglas-fir an in-vitro model system was employed. In this approach, differentiation processes were monitored by tracking wood fiber cell wall development (primary to secondary cell wall) using polarized microscopy imaging. To analyze proteins expressed at various stages of in-vitro tracheids formation several methods of protein extraction, purification and separation were investigated and a phenol-based approach was selected for subsequent studies. Protein expressed in various samples (in-vitro and in-vivo collected specimens) were then identified using a proteomics approach utilizing reverse-phase liquid chromatography coupled to tandem mass spectrometry.;Results: Developed in-vitro model system mimics cyto-differentiation process of non-differentiated Douglas-fir callus cells into mature wood fibers as proved by tracking cell wall development under polarized microscopy. Furthermore, phenol-based protocol was shown to be the most appropriate for the protein extraction from recalcitrant wood tissues. A subsequent proteomic analysis revealed proteins expressed at early and later stages of Douglas-fir wood fiber development.;Conclusions: Conducted analysis reviled the presence of proteomes sets at different stages of wood fiber development. The earliest tested phase was enriched with up regulated proteins related to cell proliferation and growth. During subsequent fiber maturation those proteins were replaced by peptides responsible for cell wall rebuilding and synthesis as an indication of advanced differentiation process.