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

17 th International Congress on Nitrogen Fixation
Fremantle, Western Australia
27 November – 1 December 2011
Title Proteomic profile of the soybean symbiosome membrane
Authors Clarke, V.C. 1 , Loughlin, P.C. 1 , Taylor, N.L. 2 , Millar, A.H. 2 , Day, D.A. 3 and Smith, P.M.C. 1
Poster Board Number 14
1 School of Biological Sciences, The University of Sydney, NSW, Australia; 2 ARC Center of
Excellence in Plant Energy Biology, The University of Western Australia, WA, Australia;
3 Flinders University, SA, Australia.
Symbiotic nitrogen fixation in legumes is characterised by the formation of a novel root organ called the nodule,
within which unique organelle-like structures termed symbiosomes develop. Free living rhizobia are engulfed
within plant cells and are surrounded by a membrane of plant origin termed the symbiosome membrane (SM). It
is this membrane that regulates the movement of solutes from plant to bacteroid (the symbiotic form of the
rhizobium) and vice versa. The SM is a unique structure containing an array of plant-derived proteins through
which the plant can regulate the symbiosis. Previous attempts to characterise the protein complement of this
membrane have been hindered by its hydrophobic nature and the absence of a complete genome for reference.
In this study, SM was isolated from mature nitrogen-fixing soybean root nodules and analysed using shotgun
proteomic techniques. The recent release of the complete soybean genome has allowed for the identification of
peptide sequences. Our proteomic analysis of soybean SM has identified eighty putative SM-localised proteins,
including ten previously localised to the SM such as nodulin-26, an aquaporin. We have focused on two classes
of proteins identified in the proteomics for further studies: amino acid transporters and ABC family transporters.
Gene expression of these candidates has been confirmed by qRT-PCR and is specific to nodule tissue.
Expression profiles across nodule development have also been completed with all candidates showing
increasing gene expression levels correlated with the onset of nitrogen fixation. This suggests these genes may
have a role in maintaining the mature symbiosis, consistent with what would be expected of a SM transporter.
amiRNA silencing vectors have been constructed to reduce expression of candidates in nodules. Further
localisation and functional studies are planned for these candidates with the aim of characterising novel
transporters on the soybean SM.
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2011