A Biosensor-Based Assay (GlnLux-Agar) Shows Defoliation Triggers Rapid Release of Glutamine from Nodules and Young Roots of Forage Legumes

Forage legumes experience defoliation from grazing and injury in both natural and agricultural ecosystems. Defoliation induces rhizodeposition of nitrogen (N) compounds from root systems that can feed microbes and plants that depend on the rhizosphere. The literature suggests that N exudates are primarily released from root tips, and those from legume nodules are released via slow nodule decomposition. However, the early timing and precise locations of N release postdefoliation are poorly characterized. The objectives of this study were to directly image tissue-specific N exudation sites in forage legumes, specifically for glutamine, and to do so at early time points post-defoliation. Glutamine is the primary assimilate of symbiotic nitrogen fixation in nodules and a key transport form of fixed N in amide-exporting legumes. Three amide-exporting forages, alfalfa (Medicago sativa), red clover (Trifolium pretense), and white clover (Trifolium repens), were defoliated or not, and placed on agar embedded with whole cell biosensor cells (GlnLux) that detect glutamine. There were two unexpected findings. First, Gln release occurred rapidly, starting within 2 h postdefoliation, depleting rapidly. Second, the sources of early Gln release were primarily nodules in addition to the expected young lateral roots/root tips. Lux quantification statistically confirmed the key findings. These observations suggest that N exudate release should be added to the list of defoliation stress early responses in nodules, and may have implications for our understanding of how defoliation impacts the rhizosphere microbiome. Furthermore, GlnLux-agar imaging represents a new assay to explore the proposed but yet unknown mechanisms underlying organic N exudation in plants.