Investigating biological nitrogen fixation via single-cell transcriptomics.

The extensive use of nitrogen fertilizers has detrimental environmental consequences, and it is fundamental for society to explore sustainable alternatives. One promising avenue is engineering root nodule symbiosis, a naturally occurring process in certain plant species within the nitrogen-fixing clade, into non-leguminous crops. Advancements in single-cell transcriptomics provide unprecedented opportunities to dissect the molecular mechanisms underlying root nodule symbiosis at the cellular level. This review summarizes key findings from single-cell studies in Medicago truncatula, Lotus japonicus, and Glycine max. We highlight how these studies address fundamental questions about the development of root nodule symbiosis, including the following findings: Single-cell transcriptomics has revealed a conserved transcriptional program in root hair and cortical cells during rhizobial infection, suggesting a common infection pathway across legume species. Characterization of determinate and indeterminate nodules using single-cell technologies supports the compartmentalization of nitrogen fixation, assimilation, and transport into distinct cell populations. Single-cell transcriptomics data has enabled the identification of novel root nodule symbiosis genes and provided new approaches for prioritizing candidate genes for functional characterization. Trajectory inference and RNA velocity analyses of single-cell transcriptomics data have allowed the reconstruction of cellular lineages and dynamic transcriptional states during root nodule symbiosis.