Project description:Objectives: to characterize and to better understand differences at a protein level in embryonic and non-embryogenic tissues of embryonal masses in Douglas-fir. In Europe, Douglas-fir is a major species for reforestation with increasing demand for its wood. Harvested stems provide timber of outstanding wood quality, mechanical properties and durability. Commercial Douglas-fir plantations in France are limited by the ability to produce seed from the latest breeding developments. Somatic embryogenesis is considered a promising biotechnology for large-scale clonal propagation of forest trees, due to the high multiplication rates it can provide. Moreover, embryogenic cultures are amenable to both cryogenic storage for long-term preservation of genetic resources and genetic engineering (including genome editing) for functional characterization of genes expressed during embryogenesis. In conifers, embryogenic cultures take the form of embryonal mass made up of early differentiated cells forming immature somatic embryos that proliferate via cleavage polyembryony. In Douglas-fir embryogenic lines consisting in embryonal mass have been compared to non-embryogenic callus during their proliferation. Comparison of proteomes (free-gel proteomics) of embryonal mass vs non-embryogenic callus were performed.
Project description:Local adaptation and phenotypic plasticity are important components of plant responses to variations in environmental conditions. While local adaptation has been widely studied in trees, little is known about plasticity of gene expression in adult trees in response to ever-changing environmental conditions in natural habitats. Here we investigate plasticity of gene expression in needle tissue between two douglas-fir provenances represented by 25 adult trees using deep RNA sequencing (RNA-Seq). Using linear mixed models, we investigated the effect of temperature, soil water availability and photoperiod on the abundance of 59189 detected transcripts. Expression of more than 80% of all identified transcripts revealed a response to variations in environmental conditions in the field. GO term overrepresentation analysis revealed gene expression responses to temperature, soil water availability and photoperiod that are highly conserved among many plant taxa. However, expression differences between the two Douglas-fir provenances were rather small compared to the expression differences observed between individual trees. Although the effect of environment on global transcript expression was high, the observed genotype by environment (GxE) interaction of gene expression was surprisingly low, since only 21 of all detected transcripts showed a GxE interaction.
Project description:First Douglas fir proteomes by nLC-MS/MS from 12 different organs : root, stem, xylem, needle, bud, female and male flowers, immature and mature seed, immature and mature somatic embryos and callus.
Project description:We report sequencing the transcriptome of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) during spring bud burst. Samples from needle tissue of two half-sibs from two families each, nested within three seed sources, were sequenced (total of six families and 12 individuals). Samples were taken at seven time points with the failure of one library, resulting in 83 total sequenced samples. Three time points were used for transcriptome assembly and 4 time point were analyzed for differential expression across time and within seed sources.
Project description:We developed a transcriptome resource for Douglas-fir covering key developmental stages of megagametophytes over time: prefertilization, fertilization, embryogenesis, and early, unfertilized abortion. Extracted RNA was sequenced using large-scale sequencing and reads were assembled to generate a de novo reference transcriptome of 105,505 predicted high-confidence transcripts. Expression levels were estimated based on alignment of the original reads to the reference.
Project description: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 to induce differentiation of callus into tracheary elements (fibers) and to monitor xylogenesisin-vitro by a proteomics approach. Two proteomes were being compared, from an early and late stage during the fiber differentiation process. After 18 weeks in a differentiating medium 80% elongated cells and 20% of cells with advanced spiral thickening were found indicating full wood fiber differentiation. Based on 2D electrophoresis, mass spectrometric, and data analyses, it was shown that nondifferentiated callus (early stage of development) expressed proteins related to protein metabolism, cellular energy and primary cell wall metabolism. At the same time, actively differentiating wood fibers (late stage of development) expressed proteins responsible for housekeeping and stress response, but mostly proteins involved in cell wall polysaccharides biosynthesis.
Project description:We developed a transcriptome resource for Douglas-fir covering key developmental stages of megagametophytes over time: prefertilization, fertilization, embryogenesis, and early, unfertilized abortion. Extracted RNA was sequenced using large-scale sequencing and reads were assembled to generate a de novo reference transcriptome of 105,505 predicted high-confidence transcripts. Expression levels were estimated based on alignment of the original reads to the reference. 200â400 megagametophytes were dissected and pooled per sample on four dates from either pollinated or unpollinated cones: June 10, June 22, June 30, and July 6 2011. These dates coincided with key events in seed development: corrosion cavity formation, fertilization, embryogenesis, or the early stages of abortion in the unpollinated treatment. Sporophytic tissue (i.e. cone bracts and cone scales) were added for comparison. PolyA RNA was used for Illumina sequencing.