IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

17 th International Congress on Nitrogen Fixation
Fremantle, Western Australia
27 November – 1 December 2011
Session Details: Tuesday 29 November 2011
Concurrent Session 4 – Applications of New Technologies
1100 - 1230
Authors: David Chiasson 1 , Patrick Loughlin 2 , Elena Fedorova 3 , Ton Bisseling 3 and Brent N.
Kaiser 1
1 School of Agriculture, Food, and Wine. The University of Adelaide, Urrbrae, SA, 5064,
Australia.
2 School of Biological Sciences, University of Sydney, Sydney New South Wales 2006,
Australia.
3 Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University,
Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.
Presentation Title: The use of heterologous expression systems to characterise the function of the soybean
transcription factor GmSAT1
Presentation Time: 1140 - 1200
GmSAT1 (Glycine max Symbiotic Ammonium Transporter) was originally isolated in a screen for
complementation of ammonium transport in the yeast mutant strain 26972c (Kaiser et al 1998). GmSAT1 has
been localized to both the peribacteroid membrane (PBM) and the nucleus in soybean nodules by Western
blotting and immunogold labeling. Recent work has revealed that GmSAT1 is critical for nodule organogenesis.
Silencing GmSAT1 by RNA interference results in a reduced number of nodules, and a Fix- phenotype.
Although the in planta function of GmSAT1 has not yet been elucidated, the use of heterologous systems has
given us insight into a very dynamic protein. We have expressed GmSAT1 in yeast and onion epidermal peels.
Expression in yeast has allowed us to confirm that GmSAT1 is indeed a transcription factor. GmSAT1 directly
activates the transcription of a novel yeast ammonium channel (termed AMF) and binds the AMF promoter in
vitro. Expression of GFP:GmSAT1 in yeast shows localization to punctate vesicles, the plasma membrane, and
the nucleus. In soybean we have recently discovered new transcripts from the GmSAT1 locus. One transcript is
an alternatively-spliced version of the original clone, and encodes for a nodule-enhanced N-terminal signal
peptide. We have bombarded onion epidermal peels with various GFP-fusions of GmSAT1. These experiments
have allowed us to determine the membrane-associated region of GmSAT1 and assign a localization function to
the signal peptide. Lastly, we have made GFP fusions of both GmSAT1 orthologues from Medicago truncatula
(MtSAT1 and MtSAT2). Confocal microscopy studies show that, similar to GmSAT1 in yeast and onions,
MtSAT1 and MtSAT2 are localized to punctate vesicles and the nucleus. We conclude that GmSAT1 is a
membrane-associated transcription factor.
Kaiser, BN, Finnegan, PM, Tyerman, SD, Whitehead, LF, Bergersen, FJ, Day, DA, and Udvardi, MK (1998).
Characterization of an Ammonium Transport Protein from the Peribacteroid Membrane of Soybean Nodules.
Science 281:1202-1205.
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2011