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The motivation of this work was to produce novel analytical techniques capable of probing the physical properties of the cell surface. Many researchers have used supported lipid bilayers (SLBs) as models to study the structure and function of the cell membrane. The complexity of these models is consistently increasing in order to better understand the myriad of physiologically relevant processes regulated by this surface. In order to aid researchers in studying such phenomenon, the following contributions were made. To manipulate components within the cell membrane, an electrophoretic flow cell was designed which can be used as a probe to study the effect of electrical fields on charged membrane components and for the separation of these components. This devise allows for the strict control of pH and ionic strength as species are observed in real-time using fluorescence microscopy. Additionally, advancements have been made to the production of patterned heterogeneous SLBs for use in separations and to probe the interactions of membrane components. The methodology to couple SLB separations and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) imaging was devised. This technology allows for the label-free mapping of the SLB surface post electrophoresis in order to observe naturally occurring species unperturbed by the addition of extrinsic tags. The final contribution, and perhaps the greatest, is the development of a procedure to create highly mobile SLBs from native membranes. These surfaces have vast potential in that they are no longer simple models of the cell surface, they are in fact the actual cell surface made planar. This advancement will be of great use to biophysicists and biochemists interested in using surface specific analytical methods to better understand physiological processes. These highly mobile native membrane surfaces have been coupled with the SLB electrophoresis technology to separate discrete bands of lipids and proteins, a proof of principle that will hopefully be further developed into a standard method for membrane proteomic studies. Collectively the tools and methodologies described herein show great potential in allowing researchers to further add to mankind’s understanding of the cellular membrane.

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Symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. Within nodule cells, N-fixing rhizobia are surrounded by plant-derived symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N2-fixing legumes have a higher requirement for P than legumes grown on mineral N. First, I investigated the impact of P deprivation on wild-type Medicago truncatula plants. My observations that plants had impaired SNF activity, reduced growth, and accumulated less phosphate in P-deficient tissues (leaves, roots and nodules) is consistent with those of similar previous studies. Galactolipids decreased with increase in phospholipids in all P-starved organs. Matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI-MSI) of phosphatidylcholine (PC) species in nodules showed that under low P environments distributions of some PC species changed, indicating that membrane lipid remodeling during P stress is not uniform across the nodule. Secondly, a metabolomics study was carried out to test the alterations in the metabolic profile of the nodules in P-stress. GC-MS based untargeted metabolomics showed increased levels of amino acids and sugars and decline in amounts of organic acids in P deprived nodules. Subsequently, LC-MS/MS was used to quantify these compounds including phosphorylated metabolites in whole plant. My findings showed strong drop in levels of organic acids and phosphorylated compounds in P deprived leaves with moderate reduction in P deprived roots and nodules. Moreover, sugars and amino acids were elevated in whole plant under P deprivation. Finally, the last project of my thesis involved studying the response of PDIL3 (Phosphate Deficiency-Induced LncRNA-3) a long non-coding RNA (lncRNA) mutant under severe P stress. PDIL3 is known to regulate Pi-deficiency signaling and transport in M. truncatula (Wang et al., 2017). My results confirmed that in P starvation, pdil3 plants showed better shoot growth, accumulated more phosphate in shoots, had impaired SNF and less rhizobial occupancy in nodules than WT. Subsequently, MALDI–MS imaging was used to spatially map and compare the distribution of phosphatidylcholine (PC) species in nodules of pdil3 and WT in P-replete and P-depleted conditions. Several PC species showed changes in distributions in pdil3 nodules compared to WT in both P sufficient and P deprived conditions. These data suggest that PDIL3's role is not just suppression of the Pi transporter, but it may also influence P partitioning between shoots and nodulated roots, meriting further investigation.