Oxygen sequestration by Leghemoglobin is positively regulated via its interaction with another late nodulin, Nlj16 of Lotus japonicus
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Nodulin proteins are expressed in legume root cells after infection with rhizobia. Nodulins have been classified as early and late, reflecting the developmental time points of their expression. Leghemoglobin (LegH), which is a classical example of a late nodulin, sequesters oxygen inside the nodule to protect the nitrogenase from oxygen toxicity to sustain symbiotic nitrogen fixation (SNF). Post-translational modification and/or protein–protein interaction are known to regulate activity of proteins. To elucidate the role of post-translational modification of LegH on its oxygen sequestration activity, earlier we have shown that phosphorylation at its S45 imparts most structural disruption of the porphyrin binding pocket responsible for its oxygen binding. In the present report, in an attempt to characterize the protein(s) that may interact with LegH to regulate its activity, it is demonstrated that LegH interact in vitro with Nodulin 16 of Lotus japonicus (Nlj16), another late nodulin. These two interacting proteins resulted in a bigger sized particle which shows higher diffusion coefficient as measured by dynamic light scattering. Interestingly, it was also shown that in vitro oxygen sequestration by LegH is stimulated by this interaction. Furthermore, this interaction is validated by the fact that LegH and Nlj16 could be co-immunoprecipitated from the nodule lysate. Most importantly, fluorescent immunohistochemistry of post-infected nodule sections show perceivable co-localization of these two proteins in the nodule symbiosomes. Thus, this work is a foundation for further investigation on these two interacting late nodulins as one of the plausible regulations for the oxygen sequestration by LegH during SNF.
KeywordsLate nodulin Leghemoglobin (LegH) Nodulin Lotus Japonicus 16 kDa (Nlj16) Oxygen sequestration Immunohistochemistry
Nodulin Lotus Japonicus 16 kDa
Symbiotic Nitrogen Fixation
Lotus Japonicus nodulin protein Phosphatase
Dynamic light scattering
Partial pressure of O2
We greatly appreciate the gift of Lotus japonicus late nodule (21 day old) cDNA library, L.japonicus seeds and Mesorhizobium loti (strain NZP2235) from Dr. J. Stougaard, Dept. of Molecular Biology, Aarhus University, Denmark. We purchased L. japonicus seeds from National Bioresource Project (Lotus japonicus, Glycine max) University of Miyazaki, Japan. We thank Dr. S.O Raja and Dr. Mohammad Azharuddin for technical support. This research was supported by the Centre for Modern Biology under University Potential for Excellence (UPE) and Center for Advanced Studies in Biochemistry awarded by the University Grants Commission (UGC) India. The financial assistance from Departments of Science and Technology and Biotechnology, Government of India (SR/SO/PS/82/2010; SR/SO/HS-51/2008; BT/PR 11415/BRB/10/656/2008); and Interdisciplinary Program in Life science (IPLS), Department of Biotechnology (DBT), Govt. of India also have supported this work. Amit Ghosh received his fellowship from IPLS program awarded by DBT and CSIR (SRF), Govt. of India. Kaushik Bhar was supported by research fellowship under the scheme for University Potential for Excellence (UPE).
AS conceived the study and designed the experiments. AG performed the immunohistochemistry in nodule section, co-immunoprecipitation and DLS experiments. KB performed the in vitro pull down assays. AG and KB both involved in protein purification and measurements of oxygen consumption. AS and AG analyzed the data and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.
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Conflict of interest
The authors declare that they have no conflict of interest.
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