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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, copper, zinc, and some other transition metals are essential nutrients for plants (
However, when aiming to improve plant nutrition in a sustainable manner, the role of plant-associated microorganisms, the plant microbiome, should also be contemplated (
This review intends to approach plant transition metal nutrition, contemplating how plants incorporate metals from soil either on their own or assisted by associated microorganisms (mainly arbuscular mycorrhiza), and how they are delivered to leaves, seeds, and, in the case of legumes, to endosymbiotic bacteria living within the root nodule cells. To do this, first we will describe some of the main protein families involved in metal transport and intracellular metal trafficking to have a conceptual frame to place the plethora of proteins involved in metal plant and plant-endoymbiont metal trafficking. For information on how plants protect themselves against toxic metal concentrations, please consider
Cellular metal homeostasis requires a highly precise regulation to ensure that transition elements are kept at high enough levels to carry out their biological functions, but not so high that they can catalyze the production of free radicals, or displace less abundant elements in the core of metalloproteins. This balance is achieved by the coordinated action of ferroreductases that provide the metals in the correct oxidative state, together with the transporters that move them across membranes, the small soluble metal binding molecules and proteins that ferry metals among the different transporters and to apoproteins, and metal detoxifying molecules that buffer the cell against a sudden increase in transition metal levels (
In the case of iron and copper are not translocated as a metal-chelator complex, the substrate is the reduced form (Fe2+/Cu+), rather than the more prevalent in aerobic environments oxidized state (Fe3+/Cu2+). Therefore, a mechanism must exist to reduce Fe3+/Cu2+. This process is carried out by members of the ferroreductase oxidase (FRO) family (
Once the metals are in the proper oxidative state or bound to the correct metallophore, they will be transported across the membrane by the different transition metal transporters. There are several families of them with distinct metal affinities and direction of transport. For the purposes of this review, we will classify them based on their direction of transport (into or out of the cytosol) since this will define their role in metal trafficking from soil to sink organs.
The best studied families are the ZIP, Ctr/COPT, Nramp, YSL, and MOT families. Their key features are:
MOT1 (Molybdate transporter type 1). In contrast to other transition metals, molybdenum is transported as the oxoanion molybdate. These transporters were first identified in parallel in
The best studied families are the P1b-ATPases, CDF, ferroportins, and VIT/CCC1 families. Their key features are:
Unlike alkali or alkali-earth metals, transition elements are not kept “free,” hydrated, in the cytosol (
When cytosolic metal concentrations rise above a certain level, metal binding capabilities of metallochaperones and organic molecules are overloaded. In that case, excessive metal levels have to be buffered by other means, since they could become toxic. Metallothioneins seem to be an universal solution to this problem (
The vacuole can also store other metals or other metal species. For instance, Zn-NA complexes are used to maintain cytosolic zinc homeostasis (
Transition metals are tightly bound to soil particles and have low solubility, specially in basic soils (
Bacteria can use both a reductive and a chelating strategy for metal uptake (
Plants benefit from this scenario. As bacteria acidify the surrounding soil, some of the solubilized metal can be used by the plant (
The relationship between plant and the microorganisms in its rhizosphere is dynamic. The plant microbiome can be altered to adapt to different developmental stages or to biotic and abiotic stresses (
Plants have developed a number of mechanisms to obtain these nutrients directly from soil. This has been best studied in the case of iron uptake, where two approaches (Strategies I and II) are followed (
In Strategy II, carried out by monocots, iron is not transported as Fe2+, but as a complex with mugineic acids (MA;
However, the separation of the two strategies (reductive vs. chelating) is not as straightforward as originally thought. Many Strategy I plants release in their root exudates a number of molecules that can solubilize Fe3+ and form complexes that are subsequently introduced into the plant. These include phenolics, coumarins carboxylates, and flavins (
Copper uptake from soil very likely follows similar strategies as for iron. In
Molybdenum is incorporated by plants as molybdate, instead of a cationic form. In green algae
Almost 90% of plants are able to establish an endosymbiotic relationship with fungi through their roots: the mycorrhiza (
The connection of AMF with plant transition metal nutrition has been known from very early on.
Metal uptake by mycorrhizal fungi should be quite similar to free living fungi. Genomic evidence indicates that model AMF
The mechanism of nutrient delivery from AMF to the host is hypothesized to be mediated by vacuoles that act as carriers (
Once the metal-loaded vacuole reaches the arbuscule, polyphosphate is hydrolized and then transferred to the host. As a result, the associated metals would be released into the cytosol by yet-to-be-determined metal transporters. Candidates for metal release are transporters of the Nramp, ZIP, and COPT families (
Nutrient recovery from the periarbuscular space is mediated by transporters specific of infected cells for phosphate and ammonia (
Although not as many as for arbuscular mycorrhiza, there are reports that indicate that ectomycorrhizal fungi are also able to buffer the plant from low or high metal levels (
Once the metals are incorporated from soil they are transported to sink organs where the demand is higher. Arguably, photosynthesis is the plant physiological process with the highest transition metal requirements, at least during vegetative growth (
Once the metals are into the root cortex, either through the epidermis or through AMF, they symplastically and apoplastically reach the endodermis (
More is known about how metals are released into the xylem. P-type ATPases play a role in metal extrusion into the xylem. In
Metal release from the xylem is not very well characterized. It has to go from the xylem into the mesophyll cells (
Most legumes, in addition to the shoot, have other metal sinks: the root nodules. These are differentiated root organs that develop as the result of the interaction with specific soil bacteria species generally known as rhizobia (
In the symbiosomes N2 is converted into NH4+, a process catalyzed by the enzyme nitrogenase. This enzyme represents 10% of the total bacteroid proteins and has a unique iron-molybdenum cofactor (FeMoco) that with the assistance of two other Fe-S clusters is responsible for the reduction reaction (
Metal delivery to the nodule could, theoretically be through the epidermal layer (as in roots), delivered by the vasculature (as the shoot), or use pre-existing metal reserves. Studies of metal visualization using synchrotron based X-ray fluorescence in
Once in the apoplast of Zone II, metals have to be incorporated by the cells (
In addition to citrate, NA should also be playing an important role in metal speciation in the nodule, since mutation in a NA synthase gene in
After being introduced into the rhizobia-infected cells, cytosolic metals have to be delivered to the same organelle as in a regular plant cell, and to symbiosomes. There is very little information on how this is done. It could merely rely in mass action effects and that metals are delivered to the organelle that has a bigger demand, and consequently a bigger “pull” on the metal reserves. Other possibility is a more directed way, either by establishing different pools accessible to only some acceptor, or by using different metal carrying proteins depending on its final destination. Identifying this mechanism is critical toward the current efforts to develop nitrogen fixing capabilities in non-legumes, since providing the metal cofactor in a timely manner is essential for nitrogenase assembly and function. These efforts will be greatly helped by the unequivocal identification of the proteins required for metal transport across the symbiosome. Seminal work by
Once they cross the symbiosome membrane, metals are accumulated in the peribacteroideal space, as indicated by radiotracer studies of iron (
From the bacteroideal periplasm, metals have to be transported into the cytosol. Molybdenum is introduced as a molybdate anion by the ModABC system (
Similar mechanisms of metal transport must be in place for other endosymbiotic interactions in which the microsymbiont is isolated from soil, as is the case of actinorhiza. In this sense, it has been shown that nodulated
Metal delivery to seeds and to younger leaves seem to be carried out through the phloem (
In addition to transfer from the xylem, phloem also obtains metals from senescent organs. As the plant flowers, sink organs such as leaves and nodules (in the case of legumes) senesce and their nutrients are recycled. Alterations in NA levels results in reproductive abnormalities, indicating that this molecule participates in metal delivery to the flowers (
Although NA plays an important role in phloem loading, it does not seem to be the major iron chelating agent in it (
Metals are also required for gametogenesis. Copper is needed for pollen tube development, as indicated by pollen abnormalities detected in
In the last three decades, we have gained a deep insight on what transporters are involved in root metal uptake and translocation to the shoot. We have also identified many of the metal-carrier molecules, as well as unveiled many of the complex regulatory pathways. More recently, as technology improved, the role of microbes in plant metal homeostasis is being better understood, as are the mechanisms mediating metal exchange with the endosymbionts. However, several other aspects have been insufficiently addressed. For instance, information on metal homeostasis in other, less studied, endosymbiosis is still lacking, very probably due to the difficulties of obtaining axenic cultures for some of them. More information is also required of the microbiome of plants growing under different levels of metal nutrition. In addition, although we know of multiple different metal transporters and some carrier proteins, their final destination, the identity of the metalloproteins that will use these metals, remains elusive. Consequently, we are missing a key element to better understand intracellular metal trafficking and use. At a systemic level we still need to determine which are the metal sensors, the signals that determine the plant metal nutritional levels, as well as to determine how the plant controls the shoot to root metal fluxes. This later aspect is especially important in legumes, since symbiotic nitrogen fixation is also an important metal sink and metal partitioning with leaves is critical to correctly balancing carbon and nitrogen fixation rates. Steps are being taken to tackle this question in the coming years. Improved methods for metalloproteomics are being developed, and elements involved in shoot-to-root metal transport in legumes are being unveiled to have a better understanding of metal partitioning in legumes, which together with improved metal imaging and metal speciation methods point toward obtaining a very clear picture on how plants use metals and the role that microorganisms have on plant metal homeostasis. This will allow us to select inoculants which will improve plant metal uptake, as well as cultivars with enhanced metal recovery capabilities from AMF or from senescent nodules, as well as increased delivery for symbiotic nitrogen fixation.
VE wrote the section on plant metal uptake, ÁS the section on metal transport in the nodule, and MT-J the section on arbuscular mycorrhiza and the molybdenum transport sections. MG-G outlined the manuscript, wrote the remaining sections, and put together the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to thank the members of the group “Plant Metal Homeostasis in Plant-Microbe Interactions” for their valuable input and criticism in preparing this review as well as Dr. Beatriz Jorrín for her help in preparing the figures. We would also like to apologize to those colleagues whose work, due to space limitations, we have not cited.