Quantitative proteomics of psychotrophic diazotroph in response to nitrogen deficiency and cold stress.

Effective protocols and novel biomarkers are the need of this hour to screen potential cold adapted diazotrophs for sustainable mountain agricultural plans. LC-MS/MS based gel less quantitative proteomics was employed to investigate the metabolic response of Himalayan cold adapted diazotroph Pseudomonas palleroniana N26 (JN055435) for nitrogen deficiency and cold stress. More than 5000 proteins were identified, and 125 of them showed significant difference with a 2-fold or greater change (p < .05) between normal and stress conditions, including 29 up-regulated proteins and 35 down-regulated proteins. Expression of nifA, nifL, nifH, nifB, nifD, and nifK during N2 fixing conditions reveals that nitrogenase system was successfully activated. Further, 8% of the upregulated proteins showed similarity with uncharacterized proteins of several nitrogen fixing genera which suggests their in-depth investigation. Additionally, as per earlier studies, cowN was differentially expressed under nitrogen fixing conditions; thereby, confirming its potential to be a potent biomarker for monitoring the nitrogen fixation in cold niches.

BIOLOGICAL SIGNIFICANCE: Understanding of nitrogenase expression and regulation is essential to employ potential diazotrophs under diverse ecological niches to achieve agricultural as well as environmental sustainability. The molecular mechanisms of cold adapted diazotrophy are still unaddressed. In this scenario, present study, besides characterizing diazotrophic proteins, is helpful in identifying the protein(s) or a biomarker viz. CowN to facilitate the monitoring of nitrogen fixation in cold niches. To the best of our knowledge, this is the first gel-less quantitative free-living diazotrophic proteome study using label free mass spectrometry having high mass accuracy in both MS and MS/MS scans. It enriches the diazotrophic proteome database and will complement the other "omics" technologies for improved crop protection and sustainability strategies.