Symbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron-molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family.
MtMOT1.2 is a Medicago truncatula MOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. A loss-of-function mot1.2-1 mutant showed reduced growth compared to wild-type plants when nitrogen fixation was required, but not when nitrogen was provided as nitrate. While no effect on molybdenum-dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen-fixing nodules, since genetic complementation with a wild-type MtMOT1.2 gene or molybdate-fortification of the nutrient solution, both restored wild-type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.
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Copper is an essential nutrient for symbiotic nitrogen fixation. This element is delivered by the host plant to the nodule, where membrane copper transporter would introduce it into the cell to synthesize cuproproteins.
COPT family members in model legume Medicago truncatula were identified and their expression determined. Yeast complementation assays, confocal microscopy, and phenotypical characterization of a Tnt1 insertional mutant line were carried out in the nodule-specific M. truncatula COPT family member.
Medicago truncatula genome encodes eight COPT transporters. MtCOPT1 (Medtr4g019870) is the only nodule-specific COPT gene. It is located in the plasma membrane of the differentiation, interzone and early fixation zones. Loss of MtCOPT1 function results in a copper-mitigated reduction of biomass production when the plant obtains its nitrogen exclusively from symbiotic nitrogen fixation. Mutation of MtCOPT1 results in diminished nitrogenase activity in nodules, likely an indirect effect from the loss of a copper-dependent function, such as cytochrome oxidase activity in copt1-1 bacteroids.
These data are consistent with a model in which MtCOPT1 transports copper from the apoplast into nodule cells to provide copper for essential metabolic processes associated with symbiotic nitrogen fixation.
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Rhizobium leguminosarum bv. viciae is a soil α-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.
Iron is an essential micronutrient for symbiotic nitrogen fixation in legume nodules, where it is required for the activity of bacterial nitrogenase, plant leghemoglobin, respiratory oxidases and other iron-proteins in both organisms. Iron solubility and transport within and between plant tissues is facilitated by organic chelators, such as nicotianamine and citrate. We have characterized a nodule-specific citrate transporter of the multidrug and toxic compound extrusion family, MtMATE67 of Medicago truncatula. The MtMATE67 gene was induced early during nodule development and expressed primarily in the invasion zone of mature nodules. The MtMATE67 protein was localized to the plasma membrane of nodule cells, and also the symbiosome membrane surrounding bacteroids in infected cells. In oocytes, MtMATE67 transported citrate out of cells in an iron-activated manner. Loss of MtMATE67 gene function resulted in accumulation of iron in the apoplasm of nodule cells and a substantial decrease in symbiotic nitrogen fixation and plant growth. Taken together, the results point to a primary role of MtMATE67 in citrate efflux from nodule cells in response to an iron signal. This efflux is necessary to ensure Fe(III) solubility and mobility in the apoplasm and uptake into nodule cells. Likewise, MtMATE67-mediated citrate transport into the symbiosome space would increase the solubility and availability of Fe(III) for rhizobial bacteroids.
In collaboration with Dr. C. Larue (CNRS), Dr. J. Imperial (CSIC), and Dr. D. Grolimund (SLS), the lab has recently published the characterization of MtZIP6 role in symbiotic nitrogen fixation in Plant Cell & Environment. This transporter is responsible for introducing Zn into rhizobia-infected cells.
Zinc is a micronutrient required for symbiotic nitrogen fixation. It has been proposed that in model legume Medicago truncatula, zinc is delivered in a similar fashion as iron, i.e. by the root vasculature into the nodule and released in the infection/differentiation zone. There, zinc transporters must introduce this element into rhizobia-infected cells to metallate the apoproteins that use zinc as a cofactor. MtZIP6(Medtr4g083570) is a M. truncatula Zinc-Iron Permease (ZIP) that is expressed only in roots and nodules, with the highest expression levels in the infection/differentiation zone. Immunolocalization studies indicate that it is located in the plasma membrane of rhizobia-infected cells in the nodule. Down-regulating MtZIP6 expression levels with RNAi does not result in any strong phenotype when plants are being watered with mineral nitrogen. However, these silenced plants displayed severe growth defects when they depended on nitrogen fixed by their nodules, as a consequence of the loss of 80% of their nitrogenase activity. The reduction of this activity was not the result of iron not reaching the nodule, but an indirect effect of zinc being retained in the infection/differentiation zone and not reaching the cytosol of rhizobia-infected cells. These data are consistent with a model in which MtZIP6 would be responsible for zinc uptake by rhizobia-infected nodule cells in the infection/differentiation zone.
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Molybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters. Members of the molybdate transporters family MOT1 were identified in the model legume Medicago truncatula and their expression in nodules determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of a Tnt1 insertional mutant line were carried out in the one M. truncatula MOT1 family member expressed specifically in nodules. Among the five MOT1 members present in M. truncatula genome, MtMOT1.3 is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. A mot1.3-1 knockout mutant showed an impaired growth concomitant with a reduction in nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore, mot1.3-1 plants transformed with a functional copy of MtMOT1.3 showed a wild type-like phenotype. These data are consistent with a model in which MtMOT1.3 would be responsible for introducing molybdate into nodule cells, which will be later used to synthesize functional nitrogenase.
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Plant microbiome and its manipulation herald a new era for plant biotechnology with the potential to benefit sustainable crop production. However, studies evaluating the diversity, structure and impact of the microbiota in economic important crops are still rare. Here we describe a comprehensive inventory of the structure and assemblage of the bacterial and fungal communities associated with sugarcane. Our analysis identified 23,811 bacterial OTUs and an unexpected 11,727 fungal OTUs inhabiting the endophytic and exophytic compartments of roots, shoots, and leaves. These communities originate primarily from native soil around plants and colonize plant organs in distinct patterns. The sample type is the primary driver of fungal community assemblage, and the organ compartment plays a major role in bacterial community assemblage. We identified core bacterial and fungal communities composed of less than 20% of the total microbial richness but accounting for over 90% of the total microbial relative abundance. The roots showed 89 core bacterial families, 19 of which accounted for 44% of the total relative abundance. Stalks are dominated by groups of yeasts that represent over 12% of total relative abundance. The core microbiome described here comprise groups whose biological role underlies important traits in plant growth and fermentative processes.
Transition metals such as iron, copper, zinc, or molybdenum are essential nutrients for plants. These elements are involved in almost every biological process, including photosynthesis, tolerance to biotic and abiotic stress, or symbiotic nitrogen fixation. However, plants often grow in soils with limiting metallic oligonutrient bioavailability. Consequently, to ensure the proper metal levels, plants have developed a complex metal uptake and distribution system, that not only involves the plant itself, but also its associated microorganisms. These microorganisms can simply increase metal solubility in soils and making them more accessible to the host plant, as well as induce the plant metal deficiency response, or directly deliver transition elements to cortical cells. Other, instead of providing metals, can act as metal sinks, such as endosymbiotic rhizobia in legume nodules that requires relatively large amounts to carry out nitrogen fixation. In this review, we propose to do an overview of metal transport mechanisms in the plant–microbe system, emphasizing the role of arbuscular mycorrhizal fungi and endosymbiotic rhizobia.
Iron is critical for symbiotic nitrogen fixation as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins, e.g. plant leghemoglobin and bacterial nitrogenase. MtNramp1 (Medtr3g088460) is the M. truncatula Nramp family member with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared to the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.
Transcription factors (TFs), transcription regulators (TR), and membrane transporters are major determinants of nodule development and function. TFs and TRs control genetic reprogramming, and transporters facilitate exchange of nutrients between plant and rhizobia among other things. Here, we describe the strategy of reverse genetic screening in Medicago truncatula for genes from these two functional classes. We employed well-established transcriptomic resources (Medicago gene expression atlas), a spatially resolved nodule transcriptomic data set, and the tobacco retrotransposon Tnt1 insertion mutant population for systematic selection and functional analysis of candidate genes. We also re-annotated 417 nodule-induced TF and TR genes according to IMGAG3.5v4, which is the latest release of the Medicago genome. In total, we have targeted 21 nodule-enhanced TF and TR genes, and 20 nodule-enhanced transporter genes for characterization via insertional mutagenesis. In this chapter, we emphasize the importance of TFs, TRs, and transport proteins for symbiotic nitrogen fixation (SNF) and discuss the use of the Medicago Tnt1 mutant population for studies of gene function.