Legumes such as soybeans, alfalfa or peanuts have the unique ability to associate with soil bacteria named rhizobia. This symbiosis leads to the formation of root nodules inside which the rhizobia transform air nitrogen into forms assimilable by the plant. Every year, in the United States, this symbiosis saves around $4.5 billion in fertilizer costs and prevents ecological and health issues associated with the excessive use of chemical fertilizers. The rhizobium-legume symbiosis presents a high level of species specificity which means that specific legumes only associate with specific rhizobia. This specificity mechanism is mostly controlled by the structure of LCO (Lipo-Chito-Oligosaccharide) signals produced by the rhizobia and recognized by their host plants. LCOs also mediate associations with symbiotic fungi which occur in legumes but also in many other non-legume crops such as corn, wheat and cotton. LCOs are now used by farmers as plant growth promoters for legume and non-legume crops. Genetic analysis in plants allowed the identification of LCO receptors and proteins such as HMGR1 (3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase 1) that interact with LCO receptors and are required for the establishment of symbiotic associations. This project will lead to a better understanding of the mechanisms allowing legumes to recognize symbiotic LCOs and of the downstream pathways allowing LCO receptors and HMGR1 to mediate the development of root nodules with rhizobia and associations with symbiotic fungi. In the short term, understanding these mechanisms can have a direct impact on yield improvement through a more efficient use of LCOs as plant growth promoters. This knowledge will also help improve the establishment of plant-microbe associations, with especially high impact under suboptimal and stressful field conditions. In the long term, engineering new symbiotic associations through these mechanisms may help improve the sustainability of our agriculture for food, feed and biofuel production.
Legumes have the ability to develop beneficial relationships with bacteria called rhizobia. During this interaction, these bacteria live in specialized organs on plant roots called nodules. Rhizobia provide otherwise unavailable atmospheric nitrogen to the legume plant. This symbiotic association allows farmers to grow legume crops using less synthetic nitrogen fertilizers, limiting water pollution and emission of greenhouse gases. Before rhizobia can enter the plant roots, they must be recognized by the plant through an exchange of diffusible signals. The perception of bacterial signals leads to the activation of several plant genes, allowing the rhizobia to penetrate in root cells and resulting in the formation of root nodules. Some of the genes that are required for the formation of the legume–rhizobium symbiosis are also present in non-leguminous plants, such as cereal plants like rice and maize, which do not form nodules. We call this set of genes the symbiotic signaling pathway, and while many of the components of the pathway have been identified, there are many missing links. For instance, it is unknown how the signals that are perceived at the cell membrane are transmitted to the nucleus, where they induce plant responses which allow for nodule formation. Our project focused on the characterization of an enzyme called 3-Hydroxy-3-methyl-glutaryl-coenzyme-A reductase 1 (HMGR1). This enzyme is a part pathway which synthesizes mevalonate, a precursor of isoprenoids which form pigments and fragrances in plants. We have shown that this enzyme, HMGR1, plays an important role in the legume–rhizobium symbiosis. We found that HMGR1 interacts with at least three cell membrane proteins that perceive symbiotic signals and that HMGR1 activity is directly regulated by one of them. The direct products of the HMGR1 activity, such as mevalonate, mevalonate 5-phosphate, and mevalonate 5-pyrophosphate, are sufficient to trigger plant responses typical of the legume-rhizobium symbiosis, such as the oscillation of calcium concentration in the nucleus (calcium spiking). We believe that HMGR1, through the production of mevalonate, is responsible for linking cell membrane signals to the nucleus. The mevalonate pathway is present in many plants and animals and results in the production of a wide range of metabolites. The products of this pathway are involved in many cell developmental processes but thus far have not been shown to play a role in signal transduction pathways. The implication that some of the compounds of this pathway are involved in the induction of nuclear calcium spiking is a step towards better understanding the symbiotic signal transduction pathway in plants. Because this pathway is conserved across land plants, the knowledge that is gained will help in engineering nitrogen fixation in cereal crops, leading to a reduction in the need for expensive synthetic nitrogen fertilizer and reducing the cost of food production. In addition to its applications to plant biology, our research has wider applications in animal systems and eukaryotic organisms in general. HMGR enzymes play important roles in moderating cholesterol levels, and various statin drugs have been developed to regulate the activity of HMGRs in humans in particular. Our work has identified new mechanisms of HMGR regulation that may provide new ways to regulate HMGR activity in the long term. Furthermore, we have identified a novel pathway for induction of calcium signaling, which is an important response for cellular communication in plant and animal cells. In addition to scientific advancement, this project has funded numerous scientific education activities for the general public and laboratory training for students. As a part of this project, a graduate student, a post-doctoral researcher, numerous undergraduate students, and several high school students, including those in under-represented groups, were trained in molecular biology, protein biochemistry, and microscopic techniques. The training of these students will provide a workforce for future discoveries in this and other fields. In addition to formal training, various outreach activities were conducted to educate the general public, including local farmers and school children, about the environmental and health hazards of the excessive use of synthetic fertilizers and the benefits of using nitrogen fixing symbiotic associations for environmentally friendly and safe crop production. In summary, this project has enabled us to identify a previously unknown mechanism of calcium signaling by compounds in the mevalonate pathway in plants, not only advancing our knowledge about beneficial symbiosis but possibly having widespread applications in other fields as well.
