The scaffold protein NifU plays a central role in assembling the precursor [Fe₄S₄] clusters required for nitrogenase to function. The synthesis of these precursors depends on a catalytic [Fe₂S₂] group within NifU core ferredoxin domain. Here, we show that the monothiol glutaredoxin GrxD is one of the proteins delivering this cluster to the NifU scaffold protein. Consistently, grxD mutants have reduced nitrogenase activity, the result of altered iron allocation to this enzyme due to a suboptimal [Fe₂S₂] cluster occupancy of the core NifU domain in the cell. These results also indicate the existence of additional pathways to provide NifU with its core [Fe₂S₂] group. Biochemical assays show that GrxD unidirectionally transfers [Fe₂S₂] to NifU through protein-protein interaction. This allows GrxD to restore apo-NifU functionality, enabling proper [Fe₄S₄] synthesis, and NifH activation. These findings are crucial to understand how iron is allocated to nitrogenase for biological nitrogen fixation.

• • Agencia Estatal de Investigación, https://ror.org/003x0zc53, PID2021-12460OB-100, PRE2022-101253, PRE2018-084895, SEV-2016-0672, CEX2020-000999S
  • United States Department of Energy, DOE-DE-SC0026054
  • Bill and Melinda Gates Foundation, INV-005889
  • Gates Foundation, https://ror.org/0456r8d26, INV-067006

Understanding how plants fend‐off invading microbes is essential for food security and the economy of large parts of the world. Consequently, a sustained and dedicated effort has been directed at unveiling how plants protect themselves from invading microbes. Major defense hormone signaling pathways have been characterized, the identity of many immune response‐triggering molecules as well as many of their receptors have been determined, and the mechanisms of pathogen‐host arms race are being studied. In recent years, evidence for a new layer of plant innate immunity involving transition metals has been brought forward. This would link plant metal nutrition with plant immune responses and open up possible new strategies for pathogen control involving metal fertilizers instead of pesticides. In this review, we outline our current understanding of metal‐mediated plant immune response and indicate the future avenues of exploration of this topic.

Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness¹. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules^2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.

Iron is an essential micronutrient for life. During the development of the seed, iron accumulates during embryo maturation. In Arabidopsis thaliana, iron mainly accumulates in the vacuoles of only one cell type, the cell layer that surrounds provasculature in hypocotyl and cotyledons. Iron accumulation pattern in Arabidopsis is an exception in plant phylogeny, most part of the dicot embryos accumulate iron in several cell layers including cortex and, in some cases, even in protodermis. It remains unknown how does iron reach the internal cell layers of the embryo, and in particular, the molecular mechanisms responsible of this process. Here, we use transgenic approaches to modify the iron accumulation pattern in an Arabidopsis model. Using the SDH2-3 embryo-specific promoter, we were able to express VIT1 ectopically in both a wild type background and a mutant vit1 background lacking expression of this vacuolar iron transporter. These manipulations modify the iron distribution pattern in Arabidopsis from one cell layer to several cell layers, including protodermis, cortex cells, and the endodermis. Interestingly, total seed iron content was not modified compared with the wild type, suggesting that iron distribution in embryos is not involved in the control of the total iron amount accumulated in seeds. This experimental model can be used to study the processes involved in iron distribution patterning during embryo maturation and its evolution in dicot plants.

Global climate change has already brought noticeable alterations to multiple regions of our planet. Several important steps of plant growth and development, such as embryogenesis, can be affected by environmental changes. For instance, these changes would affect how stored nutrients are used during early stages of seed germination as it transitions from a heterotrophic to autotrophic metabolism, a critical period for the seedling’s survival. In this perspective, we provide a brief description of relevant processes that occur during embryo maturation and account for nutrient accumulation, which are sensitive to environmental change. As examples of the effects associated with climate change are increased CO₂ levels and changes in temperature. During seed development, most of the nutrients stored in the seed are accumulated during the seed maturation stage. These nutrients include, depending on the plant species, carbohydrates, lipids and proteins. Regarding micronutrients, it has also been established that iron, a key micronutrient for various electron transfer processes in plant cells, accumulates during embryo maturation. Several articles have been published indicating that climate change can affect the quality of the seed, in terms of total nutritional content, but also, it may affect seed production. Here we discuss the potential effects of temperature and CO₂ increase from an embryo autonomous point of view, in an attempt to separate the maternal effects from embryonic effects.

Cu⁺-chaperones are a diverse group of proteins that allocate Cu⁺ ions to specific copper-proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper e.g. for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu⁺-chaperone encoded in the Medicago truncatula genome, with a N-terminus Atx1-like domain that can bind Cu⁺ with picomolar affinities. This gene is expressed primarily from the late infection zone to the early fixation zone, and is located in the cytosol, associated to plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction of nitrogenase activity, and a 50% reduction in copper-dependent cytochrome c oxidase activity. A subset of the copper-proteome is also affected in the mutant nodules. Many of these proteins can be pulled-down when using a Cu⁺-loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

The Azotobacter vinelandii molybdenum nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires an existing [Fe-S] cluster to form a [Mo-Fe₃-S₄] group, which acts as specific molybdenum donor during nitrogenase FeMo-co biosynthesis. Here, we show biochemical evidence supporting the role of NifU as the [Fe-S] cluster donor. Protein-protein interaction studies involving apo-NifQ and as-isolated NifU demonstrated their interaction, which was only effective when NifQ lacked its [Fe-S] cluster. Incubation of apo-NifQ with [Fe₄-S₄]-loaded NifU increased the iron content of the former, contingent to both proteins being able to interact with one another. As a result of this interaction, a [Fe₄-S₄] cluster was transferred from NifU to NifQ. In A. vinelandii, NifQ was preferentially metalated by NifU rather than by the [Fe-S] cluster scaffold protein IscU. These results indicate the necessity of co-expressing NifU and NifQ to efficiently provide molybdenum for FeMo-co biosynthesis when engineering nitrogenase in plants.

Iron is an essential nutrient for all life forms. Specialized mechanisms exist in bacteria to ensure iron uptake and its delivery to key enzymes within the cell, while preventing toxicity. Iron uptake and exchange networks must adapt to the different environmental conditions, particularly those that require the biosynthesis of multiple iron proteins, such as nitrogen fixation. In this review, we outline the mechanisms that the model diazotrophic bacterium Azotobacter vinelandii uses to ensure iron nutrition and how it adapts Fe metabolism to diazotrophic growth.

Co²⁺ induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition and “naked” iron import, which turns cell viability cumbersome. The N₂-fixating bacteria Sinorhizobium meliloti is a suitable model to determine the roles of Co²⁺-transporting Cation diffusion facilitator exporters (Co-eCDF) in Fe²⁺ homeostasis because it presents a putative member of this sub-familiy, AitP, and two specific Fe²⁺-export systems. An insertional mutant of AitP showed Co²⁺ sensitivity and accumulation but not Fe²⁺ sensitivity, despite AitP being a bona fide low affinity Fe²⁺ exporter as demonstrated by the kinetic analyses of Fe²⁺ uptake into everted membrane vesicles. Co²⁺ sensitivity was increased in double mutants lacking AitP and Fe²⁺ exporters but this did not correlate with the Co²⁺ accumulation, suggesting a concomitant Fe²⁺-dependent induced stress. Growth in presence of sub-lethal Fe²⁺ and Co²⁺ concentrations suggested that naked Fe-import might contribute to Co²⁺ toxicity. Supporting this Co²⁺ induced transcription of Fe-import system and genes associated to Fe homeostasis. Analyses of total protoporphyrin content point to Fe-S cluster attack as the major source for LIP. AitP mediated Fe²⁺-export is likely countered via a non-futile Fe²⁺-import pathway. Two lines of evidence support this: i) an increased hemin uptake in presence of Co²⁺ was observed in WT vs. AitP mutant and ii) hemin reversed the Co²⁺ sensitivity in the last. Thus simultaneous detoxification mediated by AitP, aid cells to orchestrate an Fe-S cluster salvage response avoiding the increase in the LIP caused by disassembly of Fe-S clusters or naked iron uptake.

Symbiotic nitrogen fixation carried out by the interaction between legumes and rhizobia is the main source of nitrogen in natural ecosystems and in sustainable agriculture. For the symbiosis to be viable, nutrient exchange between the partners is essential. Transition metals are among the nutrients delivered to the nitrogen-fixing bacteria within the legume root nodule cells. These elements are used as cofactors for many of the enzymes controlling nodule development and function, including nitrogenase, the only known enzyme able to convert N₂ into NH₃. In this review,we discuss the current knowledge onhow iron, zinc, copper, and molybdenum reach the nodules, how they are delivered to nodule cells, and how they are transferred to nitrogen-fixing bacteria within.