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

Abstract

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 15N(2) 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 H(2)O(2) and cultural experiments to assess conditions under which H(2)O(2) 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.

Fig. 1

a–e Tall fescue seedlings stained with DAB. a Fescue seedlings on agarose showing H₂O₂ secretion (red coloration) from roots; b and c Fescue root hair showing bacterial cells staining brown due to H₂O₂ concentration (arrows); d Fescue root hair showing bacterial cells losing rod structure and capacity to stain with aniline blue during oxidation (arrows; stained with DAB/peroxidase, then counterstained with 0.1 % aniline blue; e Seedling mesocotyl showing brown areas (arrows) that mark sites of bacterial oxidation

Fig. 2

a–d. Root tissues and bacteria stained with SYTO9 florescent nucleic acid stain. a and b Root hairs showing epiphytic swollen oxidizing bacteria (arrows) with intact bacteria in background; c Root parenchyma showing intensely staining intact bacteria (yellow arrows) and masses of oxidizing bacteria (blue arrow); d Root parenchyma showing nucleic acid rings (bulls-eye rings) around oxidizing bacteria (arrows)