Proteomic profile of the soybean symbiosome membrane

Abstract

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. Symbiosomes contain rhizobia which are surrounded by a membrane of plant origin termed the symbiosome membrane (SM). In this study, SM was isolated from mature nitrogen-fixing soybean root nodules and analysed using non-gel proteomic techniques. One-hundred and seventy-two proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and symbiosome space protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and targeting, signalling and transport were identified. Transport classes identified include sulphate, calcium, peptide/malate and metal ion transporters. Two classes of transport proteins identified in the proteomic analysis were selected for further characterisation: amino acid transporters and ABC transporters. In the symbiosis, amino acid transport across the SM may be involved in bacteroid nutrition while ABC transporters have been implicated in the import and export of metal ions across cell membranes and have been previously postulated to have a role in the symbiosis. Expression of candidate transporters is specific to nodule tissue, and correlates with the onset of nitrogen fixation in the developing nodule. Subcellular localisation was investigated with GFP-fusion constructs. Amino acid transporter gene expression reduction through amiRNA led to a decreased nitrogenase activity and changes in the ultra-structure of the nodule. A pilot yeast complementation study revealed two ABC transporter candidates are able to complement a plasma membrane iron transport-deficient yeast mutant. The present work provides a comprehensive analysis of the protein content of the soybean SM, and will be an invaluable tool for future studies on the legume:rhizobia symbiosis.