Research Projects

Leghemoglobin (LegH) in leguminous plants sequesters O2 molecules to protect rhizobial nitrogenase, an oxygen labile enzyme that catalyzes formation of NH3 from atmospheric N2 during symbiotic nitrogen fixation (SNF). On the other hand, SNF requires large number of ATP which is supplied by oxidative phosphorylation in the bacteriod. For this, a balance must be maintained between oxygen sequestration by LegH and that by rhizobial respiration during SNF. To address this, in our previous project funded by SERB (EMR/2017/004234/PS), we have investigated spatio-temporal status of phosphorylated LegH using anti-phospho-peptide antibodies. The outcome of the experiment revealed that P-LegH became detectable primarily in the infected cell membrane from 14dpi onwards while the non-phospho counterpart was seen as early as 9dpi. Moreover, P-LegH gradually gets dispersed in the cytosol of the infected cells towards later dpi with the increase of rhizobial load. Parallel observation in our laboratory demonstrates that LegH interacts with Nodulin 16 of Lotus japonicus (Nlj16) and this interaction is compromised when LegH is serine phosphorylated. Furthermore, Nlj16 and its related protein were shown to localize in the membrane of heterologous systems leading to our speculation that Nlj16 is responsible for localizing LegH to the infected cell membrane. Our preliminary observation also shows that the membrane localization of LegH is obliterated in the Nlj16 knocked-down infected root cells. Taken together, our findings led us to hypothesize that the subcellular localization of LegH during SNF in L.japonicus is partly regulated by its phosphorylation at serine residues and its interaction with Nlj16. In this project, we are proposing to elucidate the molecular regulation of the subcellular localization of LegH during SNF in L.japonicus to understand how the pO2 is maintained in symbiotic tissue for optimal N2 fixation. Objectives: To determine the amino acid residue(s) responsible for the localization of Nlj16 that interacts with LegH in the membrane of infected root cells To address objectives 1, mutants of Nlj16 will be generated using site directed mutagenesis followed by Agrobacterium mediated transformation of those mutants in L. japonicus plants. Nodules of different dpi after infection with M.loti will be examined by FHC. To elucidate the effect of expression of non-phosphorylatable (S to A) and phospho-mimic (S to D/E) variant(s) of LegH in its subcellular localization To address objectives 2, experiments similar to Objective 1 using mutants of LegH instead of Nlj16 will be done. To determine the impact of localization of LegH in the membrane of infected cells on SNF To address objective 3, expression of LegH variants (obtained from objectives 2) in L. japonicus plants (null background) will be carried out. After infection with M.loti, the transgenic plants with root nodule will be evaluated to monitor the efficacy of SNF by acetylene reduction assay.