Date of Award

5-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Daniel M. Roberts

Committee Members

Elias J. Fernandez, Gladys Alexandre, Brad M. Binder, Juan Luis Jurat-Fuentes

Abstract

During the infection and nodulation of legume roots by soil bacteria of the Rhizobiaceae family, the invading endosymbiont becomes enclosed within a specialized nitrogen-fixing organelle known as the "symbiosome". In mature nodules the host infected cells are occupied by thousands of symbiosomes, which constitute the major organelle within this specialized cell type. The symbiosome membrane is the outer boundary of this organelle which controls the transport of metabolites between the symbiont and the plant host. These transport activities include the efflux of the primary metabolic product of nitrogen fixation and the uptake of dicarboxylates as an energy source to support bacterial nitrogen fixation.

Soybean nodulin 26, a member of the aquaporin superfamily, is the major protein component of the symbiosome membrane that encloses nitrogen-fixing bacteroids in root nodules. Previous work has demonstrated that nodulin 26 facilitates the transport of water as well as other uncharged solutes such as glycerol and formamide. In addition, it is clear that the protein is a target for developmental and environmental sensitive posttranslational phosphorylation which may regulate transport activity.

The present research project provides evidence that nodulin 26 is an “aquaglycero-ammoniaporin” that is specifically localized to the symbiosome membrane, where it could play a potential role in osmoregulatory and metabolic functions in the symbiosis. First, it is shown that purified nodulin 26 reconstituted into liposomes possesses an ammonia permease activity that is favored approximately 4-fold over its aquaporin activity. Second, it is shown that nodulin 26 serves as a docking site for the ammonia assimilatory enzyme, cytosolic glutamine synthetase on the surface of the symbiosome membrane. Third, it is shown that phosphorylation of nodulin 26 exerts opposite effects on the regulation of ammonia and water transport activities. Fourth, it is demonstrated that phosphorylation of nodulin 26 in mature nitrogen-fixing nodules is tightly controlled by various environmental osmotic stimuli that regulate the rate of nitrogen-fixation as well as modulation of the oxygen diffusion barrier inside nodules. A model for how nodulin 26 phosphorylation could contribute to the regulation of these physiological processes is devised.

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