A proposed mechanism for nitrogen acquisition by grass seedlings through oxidation of symbiotic bacteria

In this paper we propose and provide evidence for a mechanism, oxidative nitrogen scavenging (ONS), whereby seedlings of some grass species may extract nitrogen from symbiotic diazotrophic bacteria through oxidation by plant-secreted reactive oxygen species (ROS). Experiments on this proposed mechanism employ tall fescue (Festuca arundinaceae) seedlings to elucidate features of the oxidative mechanism. We employed 15N2 gas assimilation experiments to demonstrate nitrogen fixation, direct microscopic visualization of bacteria on seedling surfaces to visualize the bacterial oxidation process, reactive oxygen probes to test for the presence of H2O2 and cultural experiments to assess conditions under which H2O2 is secreted by seedlings. We also made surveys of the seedlings of several grass species to assess the distribution of the phenomenon of microbial oxidation in the Poaceae. Key elements of the proposed mechanism for nitrogen acquisition in seedlings include: 1) diazotrophic bacteria are vectored on or within seeds; 2) at seed germination bacteria colonize seedling roots and shoots; 3) seedling tissues secrete ROS onto bacteria; 4) bacterial cell walls, membranes, nucleic acids, proteins and other biological molecules are oxidized; 5) nitrates and/or smaller fragments of organic nitrogen-containing molecules resulting from oxidation may be absorbed by seedling tissues and larger peptide fragments may be further processed by secreted or cell wall plant proteases until they are small enough for transport into cells. Hydrogen peroxide secretion from seedling roots and bacterial oxidation was observed in several species in subfamily Pooideae where seeds possessed adherent paleas and lemmas, but was not seen in grasses that lacked this feature or long-cultivated crop species

Histochemical Evidence for Nitrogen-Transfer Endosymbiosis in Non-Photosynthetic Cells of Leaves and Inflorescence Bracts of Angiosperms

We used light and confocal microscopy to visualize bacteria in leaf and bract cells of more than 30 species in 18 families of seed plants. Through histochemical analysis, we detected hormones (including ethylene and nitric oxide), superoxide, and nitrogenous chemicals (including nitric oxide and nitrate) around bacteria within plant cells. Bacteria were observed in epidermal cells, various filamentous and glandular trichomes, and other non-photosynthetic cells. Most notably, bacteria showing nitrate formation based on histochemical staining were present in glandular trichomes of some dicots (e.g., Humulus lupulus and Cannabis sativa). Glandular trichome chemistry is hypothesized to function to scavenge oxygen around bacteria and reduce oxidative damage to intracellular bacterial cells. Experiments to assess the differential absorption of isotopic nitrogen into plants suggest the assimilation of nitrogen into actively growing tissues of plants, where bacteria are most active and carbohydrates are more available. The leaf and bract cell endosymbiosis types outlined in this paper have not been previously reported and may be important in facilitating plant growth, development, oxidative stress resistance, and nutrient absorption into plants. It is unknown whether leaf and bract cell endosymbioses are significant in increasing the nitrogen content of plants. From the experiments that we conducted, it is impossible to know whether plant trichomes evolved specifically as organs for nitrogen fixation or if, instead, trichomes are structures in which bacteria easily colonize and where some casual nitrogen transfer may occur between bacteria and plant cells. It is likely that the endosymbioses seen in leaves and bracts are less efficient than those of root nodules of legumes in similar plants. However, the presence of endosymbioses that yield nitrate in plants could confer a reduced need for soil nitrogen and constitute increased nitrogen-use efficiency, even if the actual amount of nitrogen transferred to plant cells is small. More research is needed to evaluate the importance of nitrogen transfer within leaf and bract cells of plants