Exploring the symbiotic relationship of plants and microbes by mass spectrometry imaging

Abstract

Current practices in agriculture make use of chemical inputs including nitrogen fertilizers to provide high crop yields since nitrogen is often the limiting nutrient in field soil. However, nitrogen fertilizer use has negative impacts; such as due to extensive chemical runoff into waterways, increases in use of fossil fuel-derived energy with concomitant increases in greenhouse gas emissions, and accumulating costs correlated with fertilizer distribution. In contrast, biological nitrogen fixation (BNF), offers an alternative way to provide crops with required nitrogen. BNF is a process by which specific microbes convert nitrogen gas into ammonia, a form readily utilized by the plant. This dissertation focuses on two, symbiotic BNF systems: one, the intimate association between Bradyrhizobium japonicum and soybean and, two, the less intimate, associative BNF interaction between the model grass species Setaria viridis and the endophytic, plant growth promoting bacterium (PGPB) Herbaspirillum seropedicae. While these systems have both been studied at the level of the transcriptome and, to some degree, the proteome, much less is known as to how the metabolome (metabolites) change in response to bacterial colonization and nitrogen fixation. In both of these symbiotic associations, bacterial colonization is limited to specific sites and, therefore, relatively few plant cells are directly exposed to the bacteria. Therefore, in order to limit any dilution of signal due to non-responding tissues, we employed a mass spectroscopy imaging method, which allowed us to specifically target plant cells nearby or in direct contact with the bacteria. We applied an advanced and highthroughput technique called laser ablation electrospray ionization mass spectrometry (LAESI-MS) for in situ metabolic profiling. This reliable approach requires minimal sample preparation, while preserving the spatial information of biomolecules in their native state. The results provide information regarding those metabolic pathways that play a significant role in the symbiosis. Overall, the data demonstrate that LAESI-MS holds tremendous potential for use in further studies of plant-microbe interactions, as well as other plant processes.