Single-cell Raman spectroscopic analysis of bacteroids in soybean nodules to observe the relationship between biomolecular constituents and symbiotic nitrogen fixation activity.

Nitrogen fixation in soybean occurs as a result of symbiosis between the plant and rhizobia in the nodules. This process allows both the plant and the symbiont to acquire vital nutrition. To fully understand the symbiosis, many researchers have attempted to attain a deeper interpretation of the biomolecular behavior or enhance the nitrogen fixation activity of bacteroids. However, most studies have focused on forward and reverse genetics approaches to evaluate the contribution of a particular gene/enzyme in nitrogen fixation. Few studies have observed the bacteroids' overall biomolecular behavior in the nodules. Thus, we grew soybean plants and recorded acetylene reduction assay (ARA) results at several growth stages. Simultaneously, we analyzed the biomolecular compounds in the bacteroids in the nodules at the single-cell level by Raman microspectroscopy. Random forest regression, a machine learning method, was applied to discover the biomolecular contribution to the ARA, as it predicted ARA results with high accuracy. Polyhydroxybutyrate (PHB) biopolymer significantly contributed to predicting ARA results, suggesting its potential relevance in symbiotic nitrogen fixation in soybean. Further studies related to PHB behavior will lead to a deeper understanding of symbiotic nitrogen fixation and may help achieve better control of this process to increase crop yields.