Abstract
Legumes form a symbiosis with rhizobia in which the plant provides an energy source to the rhizobia bacteria that it uses to fix atmospheric nitrogen. This nitrogen is provided to the legume plant, allowing it to grow without the addition of nitrogen fertilizer. As part of the symbiosis, the bacteria in the infected cells of a new root organ, the nodule, are surrounded by a plant-derived membrane, the symbiosome membrane, which becomes the interface between the symbionts. Fractions containing the symbiosome membrane (SM) and material from the lumen of the symbiosome (peribacteroid space or PBS) were isolated from soybean root nodules and analyzed using nongel proteomic techniques. Bicarbonate stripping and chloroform-methanol extraction of isolated SM were used to reduce complexity of the samples and enrich for hydrophobic integral membrane proteins. One hundred and ninety-seven proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and PBS protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and degradation, membrane trafficking, and solute transport were identified. These included a number of proteins previously localized to the SM, such as aquaglyceroporin nodulin 26, sulfate transporters, remorin, and Rab7 homologs. Among the proteome were a number of putative transporters for compounds such as sulfate, calcium, hydrogen ions, peptide/dicarboxylate, and nitrate, as well as transporters for which the substrate is not easy to predict. Analysis of the promoter activity for six genes encoding putative SM proteins showed nodule specific expression, with five showing expression only in infected cells. Localization of two proteins was confirmed using GFP-fusion experiments. The data have been deposited to the ProteomeXchange with identifier PXD001132. This proteome will provide a rich resource for the study of the legume-rhizobium symbiosis.
Highlights
From the ‡University of Sydney, School of Biological Sciences, Sydney Australia; §Flinders University, School of Biological Sciences, Adelaide Australia
It was not observed in the peribacteroid space (PBS) or symbiosome membrane (SM) peripheral fractions but a weak signal was detected in the total nodule preparation
A small number of nodulin 26 peptides were detected in peripheral samples by LCMS/MS, suggesting the proteomic analysis is more sensitive than Western blot analysis
Summary
Plant Growth and Protein Isolation—Soybeans (G. max cv. Stephens) were grown under natural light extended to 16 h day length with incandescent lighting in a temperature controlled glasshouse (26 °C day/20 °C night). To reduce the complexity of the SM preparations by further fractionation and to enhance the collection of more hydrophobic proteins, chloroform-methanol extraction was performed on a subsequent set of four biological replicates. These four biological replicates were used to generate PBS samples. Western Blot Analysis—Ten micrograms of total nodule protein, nodule microsomal, SM (bicarbonate stripped and chloroform-methanol fractions), SM peripheral and PBS samples were separated by SDS-PAGE using Bio-Rad Mini-PROTEAN gel equipment. Three technical replicates were prepared for each sample, with multiple biological replicates analyzed for each sample type (sodium bicarbonate stripped SM: three biological replicates, C:M extracted SM: four biological replicates, SM Peripheral: two biological replicates and PBS: three biological replicates).
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