Ethylene is integrated into the nitric oxide regulation of Arabidopsis somatic embryogenesis

The study confirms the role of the two Arabidopsis hemoglobin genes (Glb1 and Glb2) during somatic embryogenesis and proposes the involvement of ethylene in the regulation of embryo development. Suppression of both Glb1 and Glb2 results in accumulation of nitric oxide (NO) and a different embryogenic response. Compared to WT tissue, down-regulation of Glb1 (Glb1 RNAi line) compromises the embryogenic process, while repression of Glb2 (Glb2−/− line) increases the number of embryos. These differences were ascribed to the differential accumulation of NO in the two lines, as Glb1 is a more effective NO scavenger compared to Glb2. A high elevation of NO level [achieved pharmacologically using the NO donor sodium nitroprusside (SNP), or genetically using the Glb1 suppressing line], activated the two ethylene biosynthetic genes 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) and 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase). Ethylene accumulation repressed embryogenesis, as shown by the decreased embryo number observed in tissue treated with the ethylene releasing agent Ethephon (ETH), as well as by the increased embryo production obtained with the two ethylene insensitive mutant lines (ein2-1 and ein3-1). A repression in ethylene level increased the expression of many auxin biosynthetic genes and favored the accumulation of the auxin indole-acetic acid (IAA) at the sites of the explants where embryogenic tissue will form. Collectively these data reveal that high levels of NO, generated by the Glb1 suppressing line, but not by the Glb2 suppressing line, might increase the level of ethylene, which represses the production of auxin. Auxin is the inductive signal required for the formation of the embryogenic tissue.