Most research in plants and other organisms has, for the sake of convenience, focused on the use of model species to identify mechanisms that are conserved throughout the whole kingdom. Nevertheless, unique features and processes such as those related to plant cell wall and fiber formation, and to wood quality, sometimes need to be studied directly in the non-model organism of interest. Such organisms, like the economically and ecologically important gymnosperm Douglas-fir (Pseudotsuga menziesii), which is one of the crucial softwood timber species in Northern America, are often difficult to investigate. High phenolic, resin, and tannin contents in the woody tissues, as well as an incompletely sequenced genome, have contributed greatly to the species' recalcitrance for molecular biology investigations. In this study, we present a complete procedure detailing protein sample preparation, separation, and proteomic analysis based on cross-species identification of Douglas-fir. Proteins from the cambial zone, mature needles, and in vitro callus were extracted, purified, and separated via 1D and 2D SDS-PAGE. One-dimensional electrophoresis coupled with ESI-MS/MS was used for cross-species protein identification in order to evaluate the potential of this approach and reveal major differences in protein profiles among tested tissues. Identified proteins were functionally and developmentally compared. The likely contribution of these proteins to the properties of the cell wall and wood is indicated and discussed.

A Douglas-fir tissue culture system was developed [1] that could be induced to differentiate into tracheary elements (fibers) making it possible to monitor xylogenesis in vitro by a proteomics approach. Two proteomes, one from an early and one from a late stage of fiber differentiation process were analyzed and compared. Obtained mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [2] with the dataset identifiers PXD001484 and DOI:10.6019/ PXD001484 [3].

The process of wood formation is of great interest to control and manipulate wood quality for economically important gymnosperms. A Douglas-fir tissue culture system was developed that could be induced to differentiate into tracheary elements (fibers) making it possible to monitor xylogenesis in vitro by a proteomics approach. Two proteomes were analyzed and compared, one from an early and one from a late stage of the fiber differentiation process. After 18 weeks in a differentiation-inducing medium, 80% of the callus cells were elongated while 20% showed advanced spiral thickening indicating full wood fiber differentiation. Based on 2D electrophoresis, MS, and data analyses (data are available via ProteomeXchange with identifier PXD001484.), it was shown that in nondifferentiated callus (representing an early stage of development), proteins related to protein metabolism, cellular energy, and primary cell wall metabolism were abundant. By comparison, in cells actively differentiating wood fibers (representing a late stage of development), proteins involved in cell wall polysaccharide biosynthesis predominated together with housekeeping and stress-associated proteins.