Detection of S-nitrosothiol and nitrosylated proteins in Arachis hypogaea functional nodule: response of the nitrogen fixing symbiont.

Debasis Maiti,Sanjay Ghosh,Tuhin Subhra Sarkar

doi:10.1371/journal.pone.0045526

Abstract

To detect the presence of NO, ROS and RNS in nodules of crack entry legumes, we used Arachis hypogaea functional nodule. The response of two cognate partner rhizobia was compared towards NO and GSNO using S. meliloti and Bradyrhizobium sp NC921001. ROS, NO, nitrosothiol and bacteroids were detected by fluorescence microscopy. Redox enzymes and thiol pools were detected biochemically. Nitrosothiols were found to be present but ROS and NO were absent in A. hypogaea nodule. A number of S-nitrosylated proteins were also detected. The total thiol pool and most of the redox enzymes were low in nodule cytosolic extract but these were found to be high in the partner microorganisms indicating partner rhizobia could protect the nodule environment against the nitrosothiols. Both S. meliloti and Bradyrhizobium sp NC921001 were found to contain GSNO reductase. Interestingly, there was a marked difference in growth pattern between S. meliloti and Bradyrhizobium sp in presence of sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Bradyrhizobium sp was found to be much more tolerant to NO donor compounds than the S. meliloti. In contrast, S. meliloti showed resistance to GSNO but was sensitive to SNP. Together our data indicate that nodule environment of crack entry legumes is different than the nodules of infection mode entry in terms of NO, ROS and RNS. Based on our biochemical characterization, we propose that exchange of redox molecules and reactive chemical species is possible between the bacteroid and nodule compartment.

Highlights

Nitric oxide (NO) is a small lipophilic molecule with immense physiological and pathological roles in biological system [1]
It is evident from our fluorimetric as well as biochemical study that RSNOs are present in a substantial amount in A. hypogaea nodules
It has been reported that RSNOs formed from low molecular weight biological thiols (L-cysteine and glutathione) are degraded in the presence of superoxide [50] producing nitrite and nitrate in the reaction mixture

Summary

Introduction

Nitric oxide (NO) is a small lipophilic molecule with immense physiological and pathological roles in biological system [1]. NO is involved in a number of physiological processes in plants [2,3]. Presence of nitric oxide synthase activity was shown in roots and nodules of Lupinus albus [4]. NO regulates the activity of numerous proteins by S- or metal nitrosylation and tyrosine nitration. S-nitrosylation of proteins are found to contribute to gene regulation [8,9,10,11], modulates phytohormonal signaling [12] and cell death [13,14,15]. Glutamine synthetase has been found to be a molecular target of nitric oxide in root nodules of Medicago truncatula where it is regulated by tyrosine nitration [16]. The biological significance of protein nitration is still not clearly understood

Methods

Results

Conclusion