In this research, we conducted histochemical, inhibitor and other experiments to evaluate the chemical interactions between intracellular bacteria and plant cells. As a result of these experiments, we hypothesize two chemical interactions between bacteria and plant cells. The first chemical interaction between endophyte and plant is initiated by microbe-produced ethylene that triggers plant cells to grow, release nutrients and produce superoxide. The superoxide combines with ethylene to form products hydrogen peroxide and carbon dioxide. In the second interaction between microbe and plant the microbe responds to plant-produced superoxide by secretion of nitric oxide to neutralize superoxide. Nitric oxide and superoxide combine to form peroxynitrite that is catalyzed by carbon dioxide to form nitrate. The two chemical interactions underlie hypothesized nutrient exchanges in which plant cells provide intracellular bacteria with fixed carbon, and bacteria provide plant cells with fixed nitrogen. As a consequence of these two interactions between endophytes and plants, plants grow and acquire nutrients from endophytes, and plants acquire enhanced oxidative stress tolerance, becoming more tolerant to abiotic and biotic stresses.

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.

Land plants can absorb soil microbes (bacterial, fungal and algal) into their cells and tissues. Plant endophytes enhance plant growth, stimulate elongation of root hairs, increase branching of roots, allow plants access to more nutrients, and stimulate oxidative stress tolerance. In the rhizophagy cycle, microbes are absorbed from soil directly into plant root cells where nutrients are extracted oxidatively, which provides nutrients to support plant growth. Early land plants lacked true roots, but possessed non-photosynthetic filaments (e.g., caulonemata, rhizoids) that may have cultivated diazotrophic bacteria within their cells as a source of nitrogen, just as bryophyte and pteridophyte rhizoids do today. Extant land plant lineages, such as bryophytes, pteridophytes, gymnosperms, and flowering plants, often produce epidermal structures (e.g., trichomes, papillae, paraphyllia, scales) on their roots, leaves, stems, or thalli; these often contain symbiotic nitrogen-fixing bacteria. Little is understood about how plants interact with soil and plant microbiomes. In this article we present novel endophytic phenomena in diverse lineages of land plants (liverworts, ferns, monocots, and eudicots) and explain how such symbiotic systems might have evolved over hundreds of millions of years. Due to these endophytic and symbiotic systems, land plants have the capability to obtain nutrients from the environment. Cultivation practices used in commercial agriculture can detract from the innate capabilities of plants to use microbes as a source of nutrients and might be harmful to plant health.