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.

To provide sustainable, sufficient, and nutritious food to a growing population is a major challenge for agriculture and the plant research community. In this respect the mineral micronutrient content of food crops deserves particular attention. Micronutrient deficiencies in cultivated soils and plants are a global problem which adversely affects crop production, plant nutritional value as well as human health and well-being. In this review, we call for awareness on the importance and relevance of micronutrients in crop production and quality. We stress the need for better nutrition in human populations with micronutrients, not only in developing but also in developed nations, and describe strategies to identify and characterize new varieties with high micronutrient content. Furthermore, we explain how an adequate nutrition of plants with micronutrients impacts metabolic functions and the capacity of plants to express tolerance mechanisms against abiotic and biotic constraints. Finally, a brief overview and a critical discussion on the current knowledge, future challenges and specific technological needs for the research on plant micronutrient homeostasis are provided. Research in this area is expected to foster the sustainable development of nutritious and healthy food crops for human consumption.

Zinc is an essential nutrient at low concentrations, but toxic at slightly higher ones. It has been proposed that hyperaccumulator plants may use the excess zinc to fend off pathogens and herbivores. However, there is little evidence of a similar response in other plants. Here we show that Arabidopsis thaliana leaves inoculated with the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM) accumulate zinc and manganese at the infection site. Zinc accumulation did not occur in a double mutant in the zinc transporters HEAVY METAL ATPASE2 and HEAVY METAL ATPASE4 (HMA2 and HMA4), which has reduced zinc translocation from roots to shoots. Consistent with a role in plant immunity, expression of HMA2 and HMA4 was up-regulated upon PcBMM inoculation, and hma2hma4 mutants were more susceptible to PcBMM infection. This phenotype was rescued upon zinc supplementation. The increased susceptibility to PcBMM infection was not due to the diminished expression of genes involved in the salicylic acid, ethylene, or jasmonate pathways since they were constitutively up-regulated in hma2hma4 plants. Our data indicate a role of zinc in resistance to PcBMM in plants containing ordinary levels of zinc. This layer of immunity runs in parallel to the already characterized defence pathways, and its removal has a direct effect on resistance to pathogens.

Legumes form a symbiosis with rhizobia that convert atmospheric nitrogen (N₂) to ammonia and provide it to the plant in return for a carbon and nutrient supply. Nodules, developed as part of the symbiosis, harbor rhizobia that are enclosed in a plant-derived symbiosome membrane (SM) to form an organelle-like structure called the symbiosome. In mature nodules exchanges between the symbionts occur across the SM. Here we characterize Yellow Stripe-like 7 (GmYSL7), a Yellow stripe-like family member localized on the SM in soybean (Glycine max) nodules. It is expressed specifically in infected cells with expression peaking soon after nitrogenase becomes active. Unlike most YSL family members, GmYSL7 does not transport metals complexed with phytosiderophores. Rather, it transports oligopeptides of between four and 12 amino acids. Silencing GmYSL7 reduces nitrogenase activity and blocks infected cell development so that symbiosomes contain only a single bacteroid. This indicates the substrate of YSL7 is required for proper nodule development, either by promoting symbiosome development directly or by preventing inhibition of development by the plant. RNAseq of nodules where GmYSL7 was silenced suggests that the plant initiates a defense response against rhizobia with genes encoding proteins involved in amino acid export downregulated and some transcripts associated with metal homeostasis altered. These changes may result from the decrease in nitrogen fixation upon GmYSL7 silencing and suggest that the peptide(s) transported by GmYSL7 monitor the functional state of the bacteroids and regulate nodule metabolism and transport processes accordingly. Further work to identify the physiological substrate for GmYSL7 will allow clarification of this role.

Yellow Stripe-Like (YSL) proteins are a family of plant transporters that are typically involved in transition metal homeostasis. Three of the four YSL clades (I, II, and IV) transport metals complexed with the non-proteinogenic amino acid nicotianamine or its derivatives. No such capability has been shown for any member of clade III but the link between these YSLs and metal homeostasis could be masked by functional redundancy. We studied the role of the clade III YSL protein MtSYL7 in Medicago truncatula nodules. MtYSL7, which encodes a plasma membrane-bound protein, is mainly expressed the pericycle and cortex cells of the root nodules. Yeast complementation assays revealed that MtSYL7 is able to transport short peptides. Medicago truncatula transposon insertion mutants with decreased expression of MtYSL7 had lower nitrogen fixation rates and showed reduced plant growth whether grown in symbiosis with rhizobia or not. YSL7 mutants accumulated more copper and iron in the nodules, which is likely to result from the increased expression of iron uptake and delivery genes in roots. Taken together, these data suggest that MtYSL7 plays an important role in the transition metal homeostasis of nodules and in symbiotic nitrogen fixation.