Regulation of metabolic events during embryo development in Norway spruce (Picea abies L. Karst)

The overall aim of this thesis was to identify and characterize metabolic and biochemical events that are involved in regulation of embryo development in Norway spruce. Embryogenesis involves coordination of multicellular patterning events which are critical for establishment of the apical-basal plan of the plant. Due to similarities with zygotic embryogenesis, the process of somatic embryogenesis (SE) provides an excellent in vitro model system for investigating the regulatory mechanisms of embryo development. Recent progress in metabolomics provides new tools for innovative approaches for elucidating the metabolic pathways present in in vitro samples. Gas chromatography coupled with time-of-flight mass spectrometry (GC/TOFMS) was used to identify important metabolic changes during development of somatic embryos in embryogenic cell lines of Norway spruce. The studied cell lines exhibited normal, aberrant and blocked development of embryos. The results of the metabolic analyses indicated that endogenous sucrose is beneficial for proliferation of proembryogenic masses (PEMs), early embryo differentiation and normal late embryo development. In contrast, aberrant late embryo formation was associated with elevated levels of endogenous fructose during embryo differentiation. A subsequent study found that embryogenic cultures of Norway spruce exhibited blocked development of embryos when cultured on medium containing fructose. Furthermore, the embryogenic cultures displayed elevated levels of protein fluorescence, protein carbonyl content, deoxyribonucleic acid (DNA) damage and alterations in antioxidant (glutathione) content. These results led to a hypothesis that the inhibitory effect of fructose on embryo development may be linked to the Maillard reaction. Assessment of the biochemical effect of carbohydrates and osmoticum on embryo development revealed that, maltose and polyethylene glycol (PEG) inhibit the germination of embryos by restricting the accumulation of sucrose, raffinose family oligosaccharides (RFOs) and late embryogenesis abundant (LEA) proteins. These compounds are important for the acquisition of desiccation tolerance. Taken together, these findings show that carbohydrates play an important role during development and germination of embryos