Elucidation of a peribacteroid membrane-bound bHLH transcription factor required for legume nitrogen fixation.

Many legumes, including soybean, are agriculturally important crop plants. Legumes are unique in their ability to form an endo-symbiosis with soil borne bacteria collectively called rhizobia, which allows the plant to access atmospheric di-nitrogen via the bacteria. The interface between the legume and differentiated, intracellular rhizobia (called bacteroids) is a plant derived membrane called the peribacteroid membrane (PBM). This membrane has a unique complement of proteins, which are required to maintain the bacteroids' environment and allow bi-directional transport of solutes. One such PBM protein from soybean is GmSAT1 (Glycine max symbiotic ammonium transporter 1) which was initially characterised as a PBM-Iocalised ammonium transporter based on its ability to complement an ammonium transport-deficient yeast strain 26972c (Kaiser et al., 1998). Subsequent research however, has suggested that GmSA T1 is not directly involved in ammonium transport (Marini et al., 2000). This project sought to shed some light on the functional role of this intriguing protein. GmSAT1 is unusual in that it has both high homology with known transcription factors of the basic Helix-Loop-Helix (bHLH) family, as well as a predicted C-terminal transmembrane domain. Conservative amino acid substitutions within the bHLH transcription factor domain of GmSAT1 completely abolished the ability of the protein to complement growth of the ammonium transport-deficient yeast 26972c on low ammonium medium. The localisation of GmSAT1 in both soybean and a yeast expression system were examined in depth using immunolocalisation, western blotting of subcellular protein fractions, and GFP fusion proteins. lmmunogold labelling of rhizobia-infected nodule cells verified the localisation of GmSATl to the PBM (Kaiser et al., 1998) and additionally the protein was localised in the nucleus. Western blotting demonstrated that GmSAT1 is present as two different size proteins in soybean nodules, with the full length protein present in the insoluble fraction and a truncated protein present in the soluble protein fraction. Biochemical evidence in yeast using a modified two-hybrid reporter system suggests that the GmSAT1 protein, either the full length protein or the N-terminal part, is localised to the nucleus. GmSAT1-GFP fusion protein was localised to small punctate bodies around the yeast cell, adjacent to the plasma membrane and in some instances co-localised with a nuclear stain, also suggesting nuclear localisation. A soybean genetic transformation protocol was developed to examine the role of GmSAT1 in the symbiosis between soybean and Bradyrhizobium japonicum through RNAi gene silencing. Results suggest that GmSAT1 is essential for normal nodule development, with GmSAT1-silenced (satl) nodules being smaller and ineffective in providing the soybean plant with sufficient fixed nitrogen. Rhizobia-infected cells in sat1 nodules were distinctive in that they retained central vacuoles and did not increase in size and consequently there were far fewer bacteroid located in these cells. Taken together, our results suggest that GmSAT1 is a membrane-bound transcription factor, located in rhizobia-infected nodule cells of soybean. Upon an as yet undetermined signal, GmSAT1 is proteolytically cleaved from the membrane and imported into the nucleus to activate gene transcription. The functional role of GmSATl in planta is yet to be determined, however silencing data suggest that it is essential for the maintenance of effective nodules.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 200