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The model legumeMedicago truncatulaestablishes a symbiosis with soil bacteria (rhizobia) that carry out symbiotic nitrogen fixation (SNF) in plant root nodules. SNF requires the exchange of nutrients between the plant and rhizobia in the nodule that occurs across a plant-derived symbiosome membrane. One iron transporter, belonging to the Vacuolar iron Transporter-Like (VTL) family, MtVTL8, has been identified as essential for bacteria survival and therefore SNF. In this work we investigated the spatial expression ofMtVTL8in nodules and addressed whether it could be functionally interchangeable with a similar nodule-expressed iron transporter, MtVTL4. Using a structural model for MtVTL8 and the previously hypothesized mechanism for iron transport in a phylogenetically-related Vacuolar Iron Transporter (VIT), EgVIT1 with known crystal structure, we identified critical amino acids and obtained their mutants. Mutants were testedin plantafor complementation of an SNF defective line and in an iron sensitive mutant yeast strain. An extended phylogenetic assessment of VTLs and VITs showed that amino acids critical for function are conserved differently in VTLs vs. VITs. Our studies showed that some amino acids are essential for iron transport leading us to suggest a model for MtVTL8 function, one that is different for other iron transporters (VITs) studied so far. This study extends the understanding of iron transport mechanisms in VTLs as well as those used in SNF. 

Abstract Background Various growth systems are available for studying plant root growth and plant–microbe interactions including hydroponics and aeroponics. Although some of these systems work well with Arabidopsis thaliana and smaller cereal model plants, they may not scale up as well for use with hundreds of plants at a time from a larger plant species. The aim of this study is to present step-by-step instructions for fabricating an aeroponic system, also called a “caisson,” that has been in use in several legume research labs studying the development of symbiotic nitrogen fixing nodules, but for which detailed directions are not currently available. The aeroponic system is reusable and is adaptable for many other types of investigations besides root nodulation. Results An aeroponic system that is affordable and reusable was adapted from a design invented by French engineer René Odorico. It consists of two main components: a modified trash can with a lid of holes and a commercially available industrial humidifier that is waterproofed with silicon sealant. The humidifier generates a mist in which plant roots grow, suspended from holes in trash can lid. Results from use of the aeroponic system have been available in the scientific community for decades; it has a record as a workhorse in the lab. Conclusions Aeroponic systems present a convenient way for researchers to grow plants for studying root systems and plant–microbe interactions in root systems. They are particularly attractive for phenotyping roots and following the progress of nodule development in legumes. Advantages include the ability to precisely control the growth medium in which the plants grow and easy observations of roots during growth. In this system, mechanical shear potentially killing microbes found in some other types of aeroponic devices is not an issue. Disadvantages of aeroponic systems include the likelihood of altered root physiology compared to root growth on soil and other solid substrates and the need to have separate aeroponic systems for comparing plant responses to different microbial strains. 

Abstract Symbiotic nitrogen (N) fixation entails successful interaction between legume hosts and rhizobia that occur in specialized organs called nodules. N-fixing legumes have a higher demand for phosphorus (P) than legumes grown on mineral N. Medicago truncatula is an important model plant for characterization of effects of P deficiency at the molecular level. Hence, a study was carried out to address the alteration in metabolite levels of M. truncatula grown aeroponically and subjected to 4 weeks of P stress. First, GC-MS-based untargeted metabolomics initially revealed changes in the metabolic profile of nodules, with increased levels of amino acids and sugars and a decline in amounts of organic acids. Subsequently, LC-MS/MS was used to quantify these compounds including phosphorylated metabolites in the whole plant. Our results showed a drastic reduction in levels of organic acids and phosphorylated compounds in –P leaves, with a moderate reduction in –P roots and nodules. Additionally, sugars and amino acids were elevated in the whole plant under P deprivation. These findings provide evidence that N fixation in M. truncatula is mediated through a N feedback mechanism that in parallel is related to carbon and P metabolism. 

Symbiotic nitrogen fixation is a complex and regulated process that takes place in root nodules of legumes and allows legumes to grow in soils that lack nitrogen. Nitrogen is mostly acquired from the soil as nitrate and its level in the soil affects nodulation and nitrogen fixation. The mechanism(s) by which legumes modulate nitrate uptake to regulate nodule symbiosis remain unclear. In Medicago truncatula , the MtNPF1.7 transporter has been shown to control nodulation, symbiosis, and root architecture. MtNPF1.7 belongs to the nitrate/peptide transporter family and is a symporter with nitrate transport driven by proton(s). In this study we combined in silico structural predictions with in planta complementation of the severely defective mtnip-1 mutant plants to understand the role of a series of distinct amino acids in the transporter’s function. Our results support hypotheses about the functional importance of the ExxE(R/K) motif including an essential role for the first glutamic acid of the motif in proton(s) and possibly substrate transport. Results reveal that Motif A, a motif conserved among major facilitator transport (MFS) proteins, is essential for function. We hypothesize that it participates in intradomain packing of transmembrane helices and stabilizing one conformation during transport. Our results also question the existence of a putative TMH4-TMH10 salt bridge. These results are discussed in the context of potential nutrient transport functions for MtNPF1.7. Our findings add to the knowledge of the mechanism of alternative conformational changes as well as symport transport in NPFs and enhance our knowledge of the mechanisms for nitrate signaling.