Changes in the glutathione and ascorbate redox state trigger growth during embryo development and meristem reactivation at germination

Abstract

In vitro embryogenesis via androgenesis and somatic embryogenesis represents an efficient propagation tool as well as a suitable model system for investigating structural, physiological, and molecular events governing embryo development. One of the major problems encountered by tissue culturists is the poor efficiency of embryos produced in vitro and their inability to regenerate viable plants, which denote sub-optimal culture conditions. Judicious alterations of the glutathione (GSH) and ascorbate (AsA) redox state have a profound effect on morphogenesis and improve the quality of the embryos by promoting a zygotic-like histodifferentiation pattern and producing well organized meristems. This approach has been investigated successfully during the development and germination of both Brassica napus (canola, an angiosperm) microspore-derived embryos (MDEs) and Picea glauca (white spruce, a conifer) somatic embryos. The imposition of a reduced glutathione environment during the early embryonic phases induces cellular proliferation and increases the number of immature embryos, possibly by promoting the synthesis of nucleotides required for energetic processes and mitotic activity. Continuation of embryo development is best conducted if the glutathione pool is experimentally switched towards an oxidized state; a condition favoring histodifferentiation and post-embryonic growth in both canola and spruce. Structural analyses showed that the oxidized glutathione environment favors the proper formation of the shoot apical meristem (SAM), which acquires a zygotic-like appearance. The apical poles of glutathione-treated embryos are well organized and display a proper expression and localization of meristem marker genes. These conditions are not found in control embryos which develop abnormal SAMs characterized by the presence of intercellular spaces and differentiation of meristematic cells. Such meristems fail to reactivate at germination resulting in embryo abortion. Physiological and molecular studies have further demonstrated that the oxidized glutathione environment induces several responses, including changes in ascorbate metabolism, abscisic acid and ethylene synthesis, as well as alterations in storage product deposition patterns. If an oxidized glutathione environment favors embryo formation, a reduced ascorbate redox state is required to promote germination and conversion, i.e. the emergence of functional shoots and roots. Specifically, a highly reduced ascorbate environment induces cell proliferation and de-novo meristem formation within those SAMs with an abnormal architecture. Overall it appears that precise changes in glutathione and ascorbate metabolism are required to ensure proper embryo formation and regeneration.