[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Nitrogen is a macronutrient that is critical for plant growth and development because it provides the building blocks of nucleic acids, proteins, chlorophyll, and energy- transfer compounds, such as ATP. Although 78% of the atmosphere is diatomic nitrogen, this form is inert and unavailable to plants due to the strong nitrogen-nitrogen triple bond. Plants can only absorb nitrogen in the forms of NH4+ or NO3-. Most of the inorganic nitrogen available to crop plants is provided through fertilizers synthesized based on the Haber-Bosch process. This process converts atmospheric nitrogen (N2) into ammonia (NH3) by a reaction with hydrogen (H2) using a metal catalyst (iron) under high temperatures (~500 [degrees]C) and high pressures (150-300 bar). Ammonia production by this method consumes a lot of energy, which is derived from burning fossil fuels. Synthetic ammonia production by the Haber-Bosch process causes losses of biodiversity through eutrophication, soil acidification and global increase in N2O atmospheric concentration, which is the third most significant greenhouse gas. An alternative approach to provide a sustainable nitrogen source to plants without causing such damage to the environment is through biological nitrogen fixation between legume species and Rhizobium bacteria. The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, specialized organs within which rhizobia convert atmospheric nitrogen into ammonia for plant consumption. In return, the legume host plants provide rhizobia with photosynthate as a carbon source for their growth. The legume - Rhizobium symbiosis is a sophisticated process that requires numerous regulators including the 20-24 nucleotide-long microRNAs which negatively regulate the expression of their target messenger RNAs. In my study, we provide two examples that demonstrate the significant role of microRNAs in the symbiotic interplay between soybean, an important legume crop, and rhizobia. In the first example, our results suggest that gma-miR319i functions as a positive regulator of nodule number during the soybean - Bradyrhizobium symbiosis by targeting the TCP33 transcription factor. Overexpression and CRISPR/cas9-mediated gene mutation of gma-miR319i increased and reduced nodule number after rhizobial inoculation, respectively. gma-miR319i and TCP33 showed an inverse expression pattern in different stages of nodule development. TCP33 modulated nodule development in a gma-miR319i dependent manner. The expression of gma-miR319i and TCP33 was differentially regulated in one soybean mutant line that exhibits a hypernodulation phenotype. In the second example, we further investigated the mechanism by which two identical microRNAs, gma-miR171o and gma-miR171q, function in modulating the spatial and temporal aspects of soybean nodulation. Although sharing the identical mature sequence, gma-miR171o and gma-miR171q genes are divergent and show unique, tissue-specific expression patterns. The expression levels of the two miRNAs are negatively correlated with that of their target genes. Ectopic expression of these miRNAs in transgenic hairy roots resulted in a significant reduction in nodule formation. Both gma-miR171o and gma-miR171q target members of the GRAS transcription factor superfamily, namely GmSCL-6 and GmNSP2. Besides those two above-mentioned examples, we were able to generate and characterize an enhancer trap insertional mutant of the NODULATION SIGNALING PATHWAY 2 (NSP2) gene which is the target gene of Gma-miR171 and also an important regulator of nodulation. Overall, our study shows the importance of microRNAs in the regulation of nitrogen-fixing symbiosis. Our results contribute to efforts to fully understand the molecular mechanisms controlling the legume - Rhizobium interaction. Our ultimate hope is that the information gained through my studies can lead to an increased utilization of biological nitrogen fixation for sustainable agriculture and environment protection.
Biological Nitrogen Fixation in the Context of Global Change
