An important building block of DNA and proteins, nitrogen (N) often limits plant growth in natural ecosystems. Still, the source of N to northern conifer forests is a long-standing ecological mystery. In these ecosystems, N accumulation in the soil and plants is much higher than expected given the known N input sources. Bacteria called endophytes, which live inside plant tissue, may provide part of the explanation. Although some forest conifers can grow in extremely nitrogen limited soil, they are not generally believed to cooperate with bacteria that can reduce, or 'fix' atmospheric N2. This project is designed to determine if endophytic bactera fix N in the aboveground tissues of forest pines. To do this, the investigators will use a method called Nanometer-Scale Secondary Ion Mass Spectrometer (NanoSIMS), an instrument suited to measure, visualize and quantify the distribution of elements and their stable isotopes within cells. The investigators will expose pine needles to the stable isotope 15N, which only makes up a fraction of naturally occurring N. NanoSIMS will then be used to visualize endophytic bacterial cells that have taken up 15N, as well as nearby plant cells containing 15N. The researchers expect this project to provide the first quantitative and direct demonstration that bacterial endophytes provision host plants with fixed atmospheric N, and identify a potential source for missing N inputs to forests. The amount of N available to forest trees affects how much carbon is stored on land and how much remains in the atmosphere, therefore, discovery of a new forest N input pathway will improve our ability to predict future climate-change. The project will provide scientific training to a diverse group of students at UC Merced, a university with a large percentage of students from underrepresented backgrounds, and to K-8 students in a rural, low-income area of California.
The goal of this project was to test the hypothesis that bacteria inside the foliage of pine trees fix atmospheric nitrogen. Nitrogen is a building block of DNA, proteins and chlorophyll, and essential to all on Earth. It is also the nutrient that limits plant growth in most ecosystems. Nitrogen is plentiful in the atmosphere, but only bacteria and archaea can access atmospheric dinitrogen and reduce it to ammonia that other organisms can use. The source of nitrogen to northern forests is a long-standing ecological mystery. In these ecosystems, nitrogen accumulation in the soil and plants is much higher than expected given the apparent nitrogen inputs. Our preliminary data suggested that bacteria inside pine foliage fix nitrogen, potentially explaining where some of the forest nitrogen comes from, and how conifers are able to grow in now nutrient environments. In this project, we have demonstrated that limber pine, a species that grows in subalpine environments throughout the Western US, hosts nitrogen-fixing bacteria inside its needles. Using DNA sequencing, we found that potentially nitrogen-fixing acetic acid bacteria are consistently present in limber pine foliage. We used the acetylene reduction assay to show that nitrogenase, the enzyme responsible for nitrogen fixation, is active inside the needles. By exposing twigs on the trees to a gas highly enriched in the heavy nitrogen isotope 15N (which is otherwise rare in the atmosphere) we demonstrated that 15N was fixed inside the foliage. Finally, we were able to visualize fixed 15N inside needles using the NanoSIMS (Nanoscale Seconday Ion Mass Spectrometer) method, which makes nanoscale maps of elemental composition (in this case the 15N/14N ratio). We saw widespread incoroporation of 15N in thinly sliced needles, surrounding bacteria cell-sized hot spots of 15N. This suggests that bacteria fix nitrogen and share with the host, which uses the fixed nitrogen as building blocks when constructing molecules. The outcomes of this award are thus 1) the first quantitative and direct demonstration that bacterial endophytes provision host plants with fixed atmospheric N, and 2) identification of a potential source for missing N inputs to forests. The broader impacts of this project are potentially very significant. Discovery of a new forest nitrogen source will improve our ability to predict future climate-change. In addition, these results could lay the foundation for efforts create nitrogen-efficient crops and provide sustainable and economical alternatives to commercial nitrogen fertilizer. The research provided training to one female first-generation college- and PhD student and two postdocs, and involved outreach efforts K-8 students in a rural, low-income area of California founded by the PIs.
