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.
