Conifer embryology : a study of polar auxin transport and WOX transcription factors

Abstract: Plants, like animals, use endogenous signaling molecules to coordinate their own physiology and development. One such molecule in plants is the hormone auxin and recent research has implicated auxin and its polar transport, together with the WOX transcription factors, in regulating embryo patterning and development in angiosperms (flowering plants), the most diverse group of land plants. No extensive investigation of this has been carried out on the more evolutionary distant gymnosperms, the other major taxa of seed plants. Thus, in this thesis I, together with my co-authors, have analyzed the regulation of embryonic pattern formation in the coniferous gymnosperm Picea abies (Norway spruce) using seed and somatic embryos. Conifers are important forestry species and knowledge of their embryology is vital for reforestration programs, where the method of somatic embryogenesis is employed for mass propagation as well as it is used as a model system for embryo development.Mature spruce embryos display a basic body plan and contain a shoot apical meristem (SAM), cotyledons, hypocotyl, embryonic root and a root apical meristem (RAM), with the meristems generating differentiated cells for organ formation. Treatment of embryos with a polar auxin transport (PAT) inhibitor produce embryos with poor SAM and, in some cases, fused cotyledons. Thus, PAT is essential for the correct patterning of conifer embryos. In angiosperms, PAT is mainly established and maintained by members of the auxin efflux facilitator PIN-FORMED (PIN) family. I isolated a PIN homologue (PaPIN1), of high abundance in conifer tissues, that is localized, together with auxin, to the epidermis of precotyledonary spruce embryos and upregulated as well as delocalized from the epidermis in early PAT inhibited embryos. Since also auxin concentration seems to decrease in the epidermis of these embryos, my data indicate that local auxin accumulation in the epidermis is mediated by PIN-dependent auxin transport.Further, I investigated WOX genes in conifers and isolated two homologues, PaWOX2 and PaWOX8/9, that are expressed throughout spruce embryo development. Their expression profile suggests involvement in cell proliferation and specification, and, indeed, they both serve as markers for conifer somatic embryogenesis. The expression of PaWOX2 and PaWOX8/9 overlap with PaPIN1 in differentiating vascular tissue (procambium) of spruce embryos and is also upregulated in early PAT inhibited embryos. Thus, PaWOX2, PaWOX8/9, and PaPIN1 may all act together in procambium differentiation. With this, I conclude that a common mechanism, involving PAT and WOX, regulate embryo pattern formation in seed plants. Results in post-embryonic tissue imply a common mechanism regulating pattern formation also here.