Manganese in Plants: From Acquisition to Subcellular Allocation

1Manganese (Mn) is an essential element in virtually all living organisms where it can fulfill two different functions: acting as an enzyme cofactor or as a metal with catalytic activity in biological clusters. In humans, Mn functions as a cofactor for a variety of enzymes, including arginase, glutamine synthetase, pyruvate carboxylase, and Mn superoxide dismutase (MnSOD). But in comparison to other essential micronutrients, such as iron (Fe) and zinc (Zn), whose deficiencies in humans are responsible for major health problems, Mn deficiency in humans is rare. However, Mn poisoning may be encountered more frequently upon overexposure to this metal causing hepatic cirrhosis, polycythemia, dystonia, and Parkinson-like symptoms. In plants, Mn is one of the 17 essential elements for growth and reproduction. It is needed in only small quantities by plants, but is ultimately as critical to growth as are the other nutrients. In photosynthetic organisms, Mn is an essential element of the metalloenzyme cluster of the oxygen-evolving complex (OEC) in photosystem II (PSII). In spite of its importance for photosynthetic activity, Mn homeostasis in plants has been poorly investigated. Nevertheless, Mn deficiency can be a serious plant nutritional disorder in soils with high pH and high partial pressure of O2 (pO2), where the bio-availability of Mn can decrease far below the level that is required for normal plant growth. Fertilization with Mn salts at soil level is often not effective, since soluble Mn (Mn2+) is rapidly converted to plant-unavailable Mn oxides, particularly in sandy alkaline soils. (In this review, we use the general term “Mn,” unless we refer to a specific oxidation state). However, it has been argued that the application of Mn fertilizers to the soil can be an effective way to alleviate Mn deficiencies, but only if soil pH is also corrected. Foliar Mn application can supply sufficient Mn to overcome Mn deficiency, but this strategy is expensive and often impractical for farmers on marginal lands. Moreover, foliar Mn sprays are only effective for a limited time period since Mn is very little mobile in the plant and does not remobilize from older leaves to Mn-deficient young leaves. On the other extreme, Mn toxicity can occur in poorly drained and in strongly acidic soils, where it is usually associated with other acidity-related soil fertility problems, such as aluminum toxicity and deficiencies of calcium (Ca), magnesium (Mg), and molybdenum (Mo).

2Depending on Mn availability, plants either need to efficiently acquire and utililize Mn under limiting conditions, or to detoxify the metal under superfluous supply. Transport processes are at the core of those adaptations. In the past, Mn transporters have been identified at the molecular level in many eukaryotic organisms. Furthermore, recent progress in Mn homeostasis has mainly focused on Arabidopsis and rice, which represent dicot and graminaceous monocot plants, respectively. Only recently, transporters involved in uptake and subcellular distribution of Mn have been characterized in a wider range of plant species. This article reviews the current knowledge of Mn uptake, translocation and subcellular distribution, as well as the functions of Mn in different plant species. Table 1 lists the Mn transporters discussed in the text.

3Mn plays a role in diverse processes of a plant’s life cycle such as photosynthesis, respiration, scavenging of reactive oxygen species (ROS), pathogen defense, and hormone signaling. In Arabidopsis, 398 enzymes are predicted to contain Mn in the metal-binding site (The UniProt Knowledgebase1). Among them, 20% showed an experimental evidence to require Mn as a cofactor. In many enzymes, Mn is interchangeable with other divalent cations such as Ca, cobalt (Co), copper (Cu), Mg, or Zn. In plants, only the OEC in PSII, MnSOD, and oxalate oxidase have been shown to require exclusively Mn.

4The most-well-studied function in plant metabolism that depends on Mn is the water-splitting reaction in PSII, which is the first step of photosynthesis. This process requires the tetra-Mn cluster Mn4O5Ca to split two water molecules into four electrons, four protons, and molecular O2. However, the delivery of Mn2+ and Ca2+ to the OEC reaction center in land plants is still under investigation. In the unicellular photosynthetic organism Synechocystis, PratA functions as an assembly factor and chaperone protein for efficient delivery of Mn to the PSII reaction core. In green plants, it is speculated that the extrinsic protein PsbP, the closest homolog to PratA, acts as a Mn carrier protein to introduce Mn into the OEC reaction center, where it subsequently stabilizes the Mn cluster in association with PsbO and PsbQ.

5Another process in plants dependent on Mn is the detoxification of ROS. In plant cells, ROS are formed mainly in chloroplasts, mitochondria, peroxisomes, and in the cytosol. Mn is a cofactor of MnSODs located in mitochondria as also in peroxisomes. In addition, oxalate oxidase is a Mn-dependent enzyme which catalyzes the oxidation of oxalate to CO2 coupled with a reduction of O2 to H2O2. Oxalate oxidase is located in the apoplast, where it is involved in pathogen defense likely by the generation of microcidal concentrations of H2O2 and by the formation of effective barriers against pathogen penetration by H2O2-mediated lignification. Oxalate oxidase activity has been identified mainly in monocot plant species including wheat, barley, and rice. Interestingly, germin-like proteins (GLPs) in Arabidopsis, which are homologous to oxalate oxidase, showed neither oxalate oxidase activity nor a Mn-dependent activation. Intriguingly, the Mn2+ cation itself may act as an antioxidant molecule. In fact, it has been shown in yeast that elevated intracellular Mn was associated with a reduction of oxidative damage in yeast cells. It is likely that Mn-phosphate (Mn-P) complexes act as antioxidants whereby the Mn speciation is altered by changes in phosphate concentrations.

6Furthermore, Mn is an important cofactor of enzymes involved in isoprenoid biosynthesis. Mn and Mg are two major cofactors of terpene synthases. Therefore, it has been proposed that different patterns of plant terpene profiles are often closely correlated with Mg/Mn ratios rather than with concentrations of each cofactor element alone in the growth media. Beside this, Mn is involved in lignin biosynthesis at two different levels: (i) Mn, besides Mg, can serve as cofactor of the phenylalanine ammonia-lyase (PAL), a key enzyme in the phenylpropanoid pathway to produce monolignols; (ii) Lignin can be synthesized from monolignols that are oxidized by Mn3+ into monolignol radicals, which can then be added to existing phenolic groups to form lignin polymers.

7Intriguingly, it has been shown that Mn can replace Mg in the active site of some enzymes of the photosynthetic pathway, including Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), changing the functional role of these enzymes. Furthermore, in vitro experiments demonstrated that Mn is an important cofactor in abscisic acid (ABA) and auxin signaling by activating PP2C phosphatases and IAA-amino acid conjugate hydrolases, respectively. Since diverse Golgi-localized glycosyl transferases require Mn for their activity, Mn is also essential for protein glycosylation and the biosynthesis of pectin and hemicellulose polymers. Other enzymes that require Mn as a cofactor are purple acid phosphatases (PAPs). Also, different decarboxylases (e.g., NAD malic enzyme) and dehydrogenases (e.g., phosphoenolpyruvate carboxylase) of the tricarboxylic acid cycle can be activated by Mn, but in many cases Mn can be replaced by Mg.

8Another Mn-dependent process lies in the deposition of cuticular waxes in leaves. In barley, it has been demonstrated that Mn deficiency may decrease the cuticular wax layer, which leads to a higher transpiration rate. Moreover, Mn deficiency caused a decrease of cuticular waxes in Arabidopsis leaves.

9In addition, Mn plays a role in such diverse processes as chloroplast development, purine and urea catabolism, phospholipid biosynthesis, Ca2+ signaling, DNA repair, or histidine biosynthesis.

10In plants, Mn deficiency often occurs as a latent disorder, without clear visual symptoms. Thus, the magnitude to which Mn deficiency affects crop yield is difficult to quantify. The critical concentration for Mn deficiency is generally below 10–20 mg.kg–1 dry weight. One of the consequences of Mn deficiency in plants is an impaired growth, leading to a decrease in biomass. This can be caused by lower numbers of chloroplasts, lower net photosynthetic efficiency, and a decrease in chlorophyll content, as well as higher susceptibility to pathogen infections, imbalance in plant water relations, and decreased tolerance to low temperatures. Mn deficiency leads to a reduced number of Mn-complexes in the PSII core, which causes the destabilization and disintegration of PSII complexes that lowers the net photosynthesis rate. Besides, the disintegration of PSII complexes directly affects the thylakoid structure and promotes chlorophyll degradation leading to the development of characteristic interveinal leaf chlorosis. Due to the low phloem mobility of Mn, typical symptoms of Mn deficiency first develop in younger leaves. Pale mottled leaves and interveinal chlorosis are the most visible symptoms of the disorder. Under severe Mn deficiency, leaves may also develop gray speck symptoms, which are characterized by brownish or necrotic spots. It has been proposed that necrotic spots are a consequence of an increase in free oxygen radicals in damaged chloroplasts and a decrease in MnSOD activity. Similar responses have been described in Chlamydomonas, where a decrease in photosynthesis and mitochondrial MnSOD function was observed under Mn-deficient conditions. However, Mn-starved Arabidopsis seedlings showed a decrease in net photosynthesis while there was no loss of MnSOD activity. This suggests that the use of the cellular Mn pool for Mn-requiring metabolic reactions under low Mn conditions is variable among photosynthetic organisms.

11In roots, an increase in the frequency of root hairs can be observed under Mn deficiency. If the deficiency becomes more severe, root tips may develop serious necrosis.

12Toxic Mn concentrations are highly dependent on plant species and genotypes. Excess Mn may be stored in vacuoles, cell walls, and distributed to different leaf tissues. Also, Mn can be chelated in Mn-P complexes in trichomes and complexed by organic acids in leaves. Therefore, it is likely that differences between plant species lie in the cellular distribution and speciation of Mn, dominated by complexes with malate or citrate. At the molecular level, excessive Mn can prevent the uptake and translocation of other essential elements such as Ca, Mg, Fe, and P, inhibit chlorophyll biosynthesis, cause a decline in the photosynthetic rate, reduce the meristematic cell division in roots by inhibiting auxin biosynthesis, and lead to an increase in the accumulation of oxidized Mn and oxidized phenolic compounds in the apoplast.

13The symptoms of Mn toxicity vary widely among plant species, with chlorotic leaves and necrotic spots as the most common symptoms. Accordingly, Mn stress in plants has been explained by two main hypotheses based on either symplastic or apoplastic processes. The symplastic hypothesis proposes that Mn toxicity acts via photo-oxidative stress in the chloroplast that causes chlorosis. Conversely, in the apoplastic hypothesis, Mn stress damage is mainly due to the accumulation of Mn oxides, oxidized phenolic compounds, and ROS, in the cell wall, leading to necrosis. Necrotic spots have thus been associated with the accumulation and oxidation of Mn and of oxidized phenolic compounds, while chlorosis has been often attributed to Fe deficiency induced by high Mn. Surprisingly, necrotic spots are high in Mn and appear first on older leaves, whereas in chlorotic areas no Mn accumulation can be observed. It is hence conceivable that the mechanism resulting in necrotic spots differs from that which causes chlorosis.

14Several lines of evidence suggest that the early target of Mn toxicity is the photosynthetic process. In fact, plants exposed to Mn excess showed a decline in net photosynthesis rate and chlorophyll content. Although in chloroplasts the occurrence of thylakoid swelling has been associated with the administration of excess Mn, the target of Mn in these photosynthetic membranes is still unclear. It has been proposed that the oxidation of Mn in chloroplasts by light-activated chlorophyll generates ROS, and thereby damages chlorophyll. At the same time, Mn may substitute Mg in chlorophyll molecules or bind to ferredoxin in the thylakoid matrix, eventually destroying the ultrastructure of chloroplasts. Moreover, the lack of physiologically active Fe would be a secondary effect of Mn toxicity, which might block chlorophyll synthesis and the correct assembly of photosystem I (PSI). Consequently, photoinhibition of PSII would likely be a late side effect of Mn exposure.

15Mn is one of the most abundant and widely distributed metals in nature and comprises about 0.1% of the Earth’s crust. The element is found in minerals, combined with other elements such as oxygen, sulfur, carbon, silicon, and chlorine. Mn can exist in 11 oxidation states, ranging from –3 to +7, but in soils, Mn is mainly present as +2 (e.g., Mn2+), +3 (e.g., Mn2O3) and +4 (e.g., MnO2). Availability of Mn to plants depends on its oxidation state: Mn2+ is the only plant-available form and can be readily transported into root cells and translocated to the shoot, whereas the oxidized species Mn(III) and Mn(IV) form insoluble oxides that rapidly sediment. Thus, Mn dynamics in soils are mostly represented by the concept of a balance between soluble Mn2+ and insoluble Mn oxides (MnOx).

16Mn deficiency in plants is particularly prevalent in alkaline soils, in which the oxidization of Mn2+ to unavailable MnOx is favored. Such soils are common in the northern part of Europe, the UK, USA, China, and in southern Australia. In addition to pH, the oxygen level (pO2) in soil and soil microorganisms are also relevant factors of Mn dynamics in soils. Beside this, root exudates can modify the Mn availability in the rhizosphere. The Mn redox status in soils involves primarily the competition of soluble highly mobile oxidants, such as molecular O2, and soluble reducing organic molecules, derived from soil organic matter and biological sources. In aerobic soils, due to the high mobility and redox potential of O2, Mn oxidation is favored rather than reduction of MnOx by microorganisms. By contrast, waterlogging causes a reduction of MnOx most likely by decreasing the O2 concentration, leading to an increase of plant-available Mn2+ in soil solution up to toxic levels.

17However, it has been proposed that the oxidation of Mn2+ in environments with abundant O2 is sluggish, particularly in the absence of biological catalysts, and that the oxidation of Mn2+ in soil is carried out predominantly by microorganisms. Indeed, microorganisms can catalyze Mn2+ oxidation in order of 103 times faster than abiotic oxidation. Some microorganisms produce enzymes which directly oxidize Mn2+, or produce extracellular superoxide leading to the production of insoluble MnOx. Conversely, other microorganisms can reduce MnOx and thereby increase Mn availability for plant uptake. Also, environmental conditions (temperature, humidity) affect soil Mn availability by modulating the activity of Mn-oxidizing microorganisms. Therefore, the population of Mn-oxidizing and Mn-reducing microorganisms is a key factor in the availability of soil Mn for plant uptake.

18Another important factor of Mn dynamics in soils is the exudation of protons (H+), carboxylates, and enzymes by plant roots. Proton exudation increases the Mn availability in the rhizosphere by exchanging Mn that is immobilized by negatively charged organic matter and clay minerals, and also by lowering the pH of alkaline soils. Mn uptake by some Mn-hyperaccumlators, such as Phytolacca species, is based on a strong rhizosphere acidification. Noteworthy, Mn availability is also increased by root exudation of carboxylates which chelate Mn and reduce Mn(IV) to Mn2+ in either acidic or alkaline soils.

19Phytate (inositol hexaphosphate) is generally the dominant form of organic P in soils and has the potential to complex Mn2+ and other divalent cations. Exudation of phytases, which catalyze the degradation of phytate thus increases the Mn availability by releasing Mn2+.

20The release of carboxylates into the rhizosphere is a mechanism for the acquisition of not only Mn, but in particular of P. Carboxylates mobilize P absorbed to Fe/Al hydroxides by ligand exchange, especially under low P availability. By contrast, elevated levels of soil-P lead to a reduction of carboxylate and phytase exudation, in turn decreasing Mn2+ acquisition. However, hydroponic experiments in barley, where most likely no Mn–P complexes were formed, have also shown a decrease in Mn uptake under elevated P supply. Hence, it has been proposed that there is a competition between Mn2+ and P during Mn uptake.

21As mentioned above, reduced Mn (Mn2+) is the only available form for plants. To maintain an optimal supply, acquisition from the rhizosphere and distribution of Mn have to be regulated. One of the most important mechanisms to regulate the acquisition from the soil is the uptake by specific transporters into root cells (Figure 1). Once Mn reaches the symplast, the main pathways for the translocation and distribution of Mn in the whole plant involve transport toward and into the xylem, transfer to the phloem, and translocation to and between the different tissues. However, most of the Mn transporters and mechanisms required for Mn loading into the xylem of the root stele, for loading Mn into the phloem, and for Mn transport into the plant cell in shoots have not been identified yet. Interestingly, the mobility of Mn in the phloem is supposed to be low, but there is evidence that a small amount of Mn may be recycled via the phloem. In fact, it has been reported that after application of 52Mn to a cut barley leaf, radioactivity was detectable in the discrimination center of the shoot, in other leaves, as also in root tips. These results suggested that 52Mn was transported by unknown transporters with low affinity.

22An intriguing property of Mn transport across membranes is that most of the proteins that transport Mn are not specific for the metal. Plant Mn2+ transporters can transport, to varying extent, other divalent cations, such as Fe2+, Zn2+, Cu2+, Cd2+, Ca2+, Co2+, and Ni2+. The physiological relevance of this low specificity needs further investigations, including the modification of specificity. For instance, in the Mn/Fe transporter AtMTP8, Fe transport activity was inhibited by introducing point mutations in different Fe-binding domains without affecting its Mn2+ transport capability.

23Diverse families of transport proteins are known to be components of the Mn homeostatic network in plants and can be classified into importers and exporters. Importers translocate Mn from the extracellular space or from internal compartments into the cytosol, whereas exporters are responsible for the exclusion of Mn out of the cytosol into intracellular compartments or into the apoplast. The Natural Resistance Associated Macrophage Protein (NRAMP) family, the Zinc-Regulated Transporter/Iron-Regulated Transporter (ZRT/IRT)-related Protein (ZIP) family, and the Yellow Stripe-Like (YSL) family have members involved in the transport of Mn2+ into the cytosol. In contrast, the Cation Diffusion Facilitator/Metal Transport Protein (CDF/MTP) family, the Vacuolar Iron Transporter (VIT) family, the Ca2+/Cation Antiporter (CaCA) superfamily, the Bivalent Cation Transporter (BICAT) family, and the P2A-type ATPase family have members involved in the transport of Mn2+ out of the cytosol. Figure 1 shows the tissue specificity and subcellular localization of Mn transport proteins in roots of Arabidopsis, rice, and other plant species. Genes encoding encoding Mn transport proteins in aerial parts of the plant species are depicted in Figure 2, and the subcellular localization of these proteins is displayed in Figure 3. The NRAMP family has been characterized in a number of species including bacteria, fungi, plants, and animals. These proteins act as metal/H+ symporters and are capable of transporting divalent metal cations (Fe2+, Mn2+, Zn2+, Cd2+, Co2+, Ni2+, Cu2+) into the cytosol, with the exceptions of OsNRAMP4 (syn. OsNrat1), that transports the trivalent cation Al3+, and OsNRAMP1, that appears to mediate also As(III) transport.

24The ZIP transporters have been found widely in bacteria, fungi, plants, and animals and are predicted to be involved in Fe2+, Zn2+, Cd2+, Co2+, Cu2+, and Mn2+ transport. They have 8 transmembrane domains (TMD) with extracellular N- and C-termini and a cytosolic histidine-rich loop. YSL transporters are related to the OligoPeptide Transporter (OPT) family and are exclusively found in plants, bacteria, fungi, and archaea. Members of the YSL family are predicted to transport metals (Mn2+, Zn2+, Cu2+, Ni2+, Cd2+, Fe2+) complexed to non-proteinogenic amino acids, such as nicotianamine (NA) or phytosiderophores (PS).

25The CDF family has members in all organisms. Most CDFs are Metal2+/H+(K+) antiporters and mediate the efflux of Zn2+, Co2+, Fe2+, Cd2+, Ni2+, and/or Mn2+. Most of them have six TMDs with histidine-rich regions at their cytosolic N- and/or C-terminus and additionally between the 4th and 5th TMD. Based on phylogenetic relationships, the CDF family can be classified, corresponding to the main transported metal, into three major subgroups: Zn-CDFs, Fe/Zn-CDFs, and Mn-CDFs.

26BICAT proteins, also denominated Photosynthesis-Affected Mutant71 (PAM71) or Chloroplast Manganese Transporter (CMT) proteins, belong to the Uncharacterized Protein Family 0016 (UPF0016), which represents a new family of cation transporters present in all eukaryotes and prokaryotes, except in Lactobacillales and Bacillales. The protein structure of the plant homologs is characterized by two clusters of three transmembrane domains separated by a central loop. BICAT proteins act as Mn2+ and Ca2+ transporters.

27CaCA transporters are present in all kingdoms of life and have a conserved core structure of ten transmembrane domains and two conserved α-repeat regions containing acidic amino acids. In particular, the Cation/Ca2+ exchanger (CCX) and the H+/Cation exchanger (CAX) families, which are both members of the CaCA superfamily, are present within all plants.

28VIT transporters are found in plants, fungi, and bacteria, but are absent in animals. Members of the VIT family in plants share a high degree of sequence similarity and most of them are capable to transport Fe2+ in addition to Mn2+, but their biological functions have been poorly investigated.

29Plant P-type Ca2+-ATPases are divided into two groups, 2A and 2B, whereby the first one does not contain an N-terminal autoinhibitory domain. Those P2A-type Ca2+-ATPases have also a role in Mn2+ transport.

30Despite the importance of Mn in plant physiology, our knowledge of systems mediating Mn uptake followed by translocation from the roots to the shoot is limited. This is mainly due to the lack of information about the expression and subcellular localization of Mn transporters in most plants. In fact, only few Mn transporters involved in uptake and root radial transport have been identified so far. Uptake of Mn2+ has been assumed to be mediated by plasma membrane Ca2+ channels, which are generally permeable to Mn2+. However, this is likely to be a minor Mn2+ uptake pathway due to its competition with Ca2+, only relevant when Mn2+ is present in high concentrations. However, in soil, Mn2+ is usually far less abundant than Ca2+. Since this assumption, few Mn transporters have been identified mainly in A. thaliana and rice. Their tissue-specific localizations in roots are shown in Figure 1A for Arabidopsis, in Figure 1B for rice, and in Figure 1C for other plant species including barley, wheat, tomato, poplar, and grapevine.

31In Arabidopsis, there is plenty of evidence that Mn2+ uptake is mainly mediated by AtNRAMP1 (Figure 1A), which is localized in the plasma membrane of epidermis and cortex cells in roots, and less in vascular tissues of young leaves. Under Mn-deficient conditions, the expression of AtNRAMP1 is moderately up-regulated, and nramp1 knockout mutants accumulate less Mn in shoots under Mn deficiency, which points to a function of this protein as a high-affinity Mn2+ uptake transporter. NRAMP1 orthologs in cacao, rapeseed, and peanut are also expressed in roots and complement a Mn uptake-deficient yeast strain, smf1, lacking a Mn transporter located in the plasma membrane. In addition, active Mn2+ uptake may be accomplished by the ZIP transporter AtIRT1, that is considered as the major Fe uptake transporter of dicots. However, AtIRT1 is not strongly selective for Fe2+, but also transports Zn2+, Cu2+, Co2+, Ni2+, and Mn2+. IRT1 orthologs expressed in roots have also been identified in tomato and trifoliate orange, but their function in Mn2+ uptake remains to be confirmed. Furthermore, two ZIP transporters expressed in the root stele are described to be involved in root-to-shoot translocation of Mn in Arabidopsis. AtZIP1 is a transporter localized to the tonoplast and probably involved in remobilizing Mn2+ from vacuoles to the cytosol in root stele cells, whereas AtZIP2 is localized to the plasma membrane and may mediate Mn2+ uptake into cells of the root stele. Other Mn2+ transporters of the AtZIP family are also expressed in roots, but their function in Mn2+ uptake and remobilization is still unknown.

32In comparison with other plant species, in rice, transporters involved in uptake, xylem loading, and root-to-shoot translocation of Mn have been functionally characterized more extensively. The first Mn2+ transporter identified in rice roots was OsNRAMP5, which contributes to Mn2+ uptake and translocation. Further investigations showed that Mn2+ uptake and translocation works in conjunction with another transporter next to OsNRAMP5, namely OsMTP9. OsNRAMP5, localized to the plasma membrane of the distal side of the exo- and endodermis was shown to be responsible for the import of Mn2+ from the soil solution (Figure 1B). Subsequently, OsMTP9, localized to the plasma membrane of the proximal side of these cell layers, mediates the export of Mn2+ into the stele. Knockout of either OsMTP9 or OsNRAMP5 significantly decreased Mn2+ uptake and root-to-shoot translocation, indicating that both transporters are responsible for transporting Mn2+ from the soil to the xylem.

33Interestingly, in barley, the plasma membrane transporter HvIRT1 is implicated in the uptake and translocation of Mn2+, but not Fe2+. HvIRT1 is constitutively expressed in cells of the epidermis and in pericycle funder cells (Figure 1C), but under Mn deficiency, its expression is extended to the entire pericycle and the cortex. In hvirt1 RNAi lines, a reduced shoot Mn concentration was observed without changes in Fe or Zn concentrations. Barley is characterized as a strategy II plant species that requires HvYSL transporters for the uptake of Fe3+ complexed with phytosiderophores (PS). Therefore, the Fe transport function of HvIRT1 has become redundant because Fe is acquired via strategy II processes. Although HvIRT1 may transport Fe in yeast, HvIRT1 plays actually a key role in uptake and root-to-shoot translocation of Mn rather than Fe. HvNRAMP5 is another Mn transporter localized to the plasma membranes of cells in root tips of barley. In contrast to HvIRT1, its expression is slightly upregulated by Fe deficiency, but not by Mn deficiency. Since HvIRT1 is up-regulated by Mn deficiency and HvNRAMP5 is constitutively expressed in roots, both transporters may play different roles. Therefore, it has been suggested that HvNRAMP5 may confer constitutive Mn2+ uptake, while HvIRT1 plays a role in Mn2+ uptake only under Mn-deficient conditions. The barley transporter HvYSL2 appears to mediate the transport of PS complexes with metals, including Mn, in the endodermis, and it may thus be involved in the transport of minerals from the cortex to the pericycle. However, further analyses of HvYSL2 are required to fully understand its biological role in barley.

34Once Mn has been absorbed by the root, it needs to be translocated to the shoot. To date, the molecular basis of xylem loading of Mn is still unclear, and there is no clear evidence of which Mn complex is required to translocate Mn to the shoot via the xylem. Nevertheless, transporters of the YSL family may be involved in Mn translocation. In fact, during senescence, Mn concentration decreased in Arabidopsis wild type leaves, whereas no change was observed in the ysl1ysl3 double mutant. Hence, it has been proposed that both AtYSL1 and AtYSL3 are putative Mn-nicotianamine (Mn2+-NA) transporters, but so far, there is no direct evidence supporting this hypothesis. In rice, Mn2+ is transferred by OsNRAMP3 from the xylem to the phloem at the basal node, followed by its distribution to young leaves, panicles and root tips. However, at high Mn availability, Mn is distributed to mature tissues. Therefore, in rice nodes OsNRAMP3 functions as a switch for Mn distribution, whereby the protein is activated or deactivated in response to fluctuating Mn concentrations (Figure 2B). Moreover, a rice YSL transporter, OsYSL2, was proposed to be implicated in long-distance transport and distribution of Mn, since it may transport Mn2+-NA, as well as Fe2+-NA complexes. Because OsYSL2 is localized in phloem companion cells, it is probably involved in phloem loading of Mn2+-NA, although its exact role in rice needs to be further investigated.

35As described above, a number of plasma membrane transporters involved in Mn2+ import into the cytosol of root cells has been characterized. In contrast, the identity of Mn2+ transporters in the plasma membrane of leaf cells remains largely elusive. Recently, it has been reported that OsNRAMP6, which is expressed in shoots, is localized to the plasma membrane (Figure 3B) and functions as Fe and Mn2+ transporter when expressed in yeast. Interestingly, OsNRAMP6 rescued, to some extent, the growth of yeast strains mutated in SMF1 (plasma membrane Mn2+ transporter) or SMF2 (Mn2+ transporter in intracellular vesicles) under Mn-limited conditions. In plants, OsNRAMP6 accumulated in vesicles in the vicinity of the plasma membrane. Whether these vesicles represent an anterograde or retrograde trafficking stage of OsNRAMP6, depending on the Mn2+ status, remains to be elucidated. Moreover, an OsNRAMP6 knock-out plant showed enhanced resistance to infection by the rice fungus M. oryzae and a reduced biomass compared to wild type plants. Therefore, it is likely that OsNRAMP6 plays a role in regulating the Mn and/or Fe contents in infected tissues which would boost the expression of defense genes. Based on the decreased root and shoot biomass of the nramp6 mutant under non-stress conditions, it was hypothesized that OsNRAMP6 functions as a Mn uptake transporter. Future studies are needed to establish the importance of OsNRAMP6 in cellular Mn2+ uptake and plant growth.

36A second mechanism for Mn uptake into leaf cells of rice may be conferred by the OsYSL6 transporter, which was described to transport Mn2+-NA complexes from the leaf apoplast to the symplast. OsYSL6 is expressed in roots and shoots, particularly in older leaves. Due to the ability of OsYSL6 to increase Mn2+ influx when expressed in the smf1 yeast mutant, it is likely localized in the plasma membrane. ysl6 mutant plants accumulate high Mn concentrations in the apoplast of shoots and exhibit symptoms of Mn toxicity. Therefore, it is likely that OsYSL6 translocates Mn2+ from the apoplast to the symplast where it is sequestered under Mn excess. However, since its expression level remains unchanged under either Mn deficiency or excess, OsYSL6 may also act as a constitutive Mn importer in leaf cells.

37Once Mn has entered a plant cell, it must be moved to the appropriate location for the adequate supply of Mn-dependent targets or for storage. Mn is present in all cellular compartments, including ER, Golgi apparatus, mitochondria, plastids, and peroxisomes, where it performs specific cellular functions (see section Functions of Manganese in Plants), whereas vacuoles can serve as a reservoir to regulate cellular Mn homeostasis. Consequently, cells contain a plethora of transporters that are responsible for the distribution of Mn to the different compartments. Figure 3 shows an overview of Mn transporters previously described in different plant species and their subcellular localization. To structure the description of these Mn transporters in the following sections, they are organized based on their demonstrated or putative subcellular localization.

38Vacuoles generally function as a primary compartment for metal internalization to avoid metal toxicity. In the case of Mn, vacuoles also serve as a temporal Mn storage pool for a proper distribution to other organelles, e.g., chloroplasts. Many transporters have been described to contribute to vacuolar Mn sequestration and unloading processes.

39In the tropical legume Stylosanthes hamata, a plant tolerant to acidic soils in which high concentrations of plant-available Mn2+ can occur, the tonoplast-localized Mn transporter ShMTP8 was identified. This protein was the first characterized member of the Mn-CDF subgroup and is involved in Mn detoxification by sequestering Mn into vacuoles. Sequence analysis of ShMTP8 showed that this protein lacks the complete N-terminal sequence and the histidine-rich loop common for members of the CDF family. When expressed in Arabidopsis, ShMTP8 conferred tolerance to Mn toxicity. In pear and tobacco, members of the Mn-CDF subclade have also been described as Mn transporters, but their subcellular localization and relevance in Mn homeostasis is unknown. In Arabidopsis, the Mn-CDF subclade of the CDF/MTP family has four members, AtMTP8 through AtMTP11. All of them were shown to transport Mn2+ in yeast, but so far, only AtMTP8 and AtMTP11 have been described in more detail (Figure 3A). AtMTP8 was characterized as Mn2+ and Fe2+ transporter localized in the tonoplast. AtMTP8 expression was specific to cells of the epidermis and the cortex in roots and strongly induced by Fe deficiency. Moreover, mtp8 mutants showed chlorosis and critically low Fe levels in shoots on media with limited Fe availability, if Mn was present in the medium. This further demonstrated a Mn-specific role of AtMTP8 during Fe limitation, which lies in the detoxification of Mn taken up by the non-specific Fe transporter AtIRT1. In accord with a function of AtMTP8 in Mn detoxification, growth of mtp8 mutants was impaired by high Mn, and AtMTP8 expression was increased under excess Mn2+ supply. Besides its role in the Fe deficiency response, AtMTP8 plays a second role during seed development. An analysis of metal localization in the embryo by μXRF tomography showed that AtMTP8 is responsible for the specific accumulation of Mn in subepidermal cells on the abaxial side of the cotyledons and in cortical cells of the hypocotyl. In mutant embryos lacking the vacuolar Fe/Mn transporter AtVIT1, AtMTP8 built up Fe hotspots in those AtMTP8-expressing cell types, suggesting that AtMTP8 transports Fe in addition to Mn. This was supported by complementation of the Fe-sensitive yeast strain ccc1. An mtp8vit1 double mutant showed a homogeneous distribution of both metals in all cell types of the embryo, demonstrating that both are the primary transporters determining Mn and Fe allocation. Mn2+ transport and vacuolar localization were also demonstrated for MTP8 orthologs in rice (Figure 3B). OsMTP8.1 was highly expressed in older leaves, and it was induced and repressed by high and low Mn2+ levels, respectively. OsMTP8.2 was expressed in roots and shoots, and it showed lower expression levels than OsMTP8.1. The mtp8.1mtp8.2 double mutant suffered from necrotic leaf blades. Since Mn concentrations were comparable to those in healthy wild type plants, it has been suggested that this phenotype was the result of an insufficient Mn sequestration in the double mutant. Unlike AtMTP8 in Arabidopsis, there is no evidence that OsMTP8.1 and OsMTP8.2 are able to transport Fe or that their transcript levels are increased upon Fe deficiency.

40VIT proteins in plants are mainly Fe transporters. AtVIT1, the most-studied VIT transporter in Arabidopsis, was identified as a vacuolar Fe transporter that is responsible for the localization of Fe primarily to the provascular strands of the embryo in seeds. The ability of AtVIT1 to transport Mn2+ was shown by metal analysis of vacuoles from the Fe-sensitive yeast strain ccc1 expressing AtVIT1. Interestingly, seeds of a vit1 mutant showed a localization of Fe that coincided with the localization of Mn in the subepidermal cells on the abaxial side of the cotyledons and that was dependent on AtMTP8, as discussed above. VIT transporters of rice, OsVIT1 and OsVIT2, not only partially rescued the Fe2+-sensitive phenotype, but also the Zn2+-sensitive phenotypes of yeast mutant strains. Similar to AtVIT1, an analysis of vacuolar metal composition of these cells showed an increased accumulation of Mn. Moreover, OsVIT1 and OsVIT2 were shown to be highly expressed in flag leaf blades and leaf sheath. Consistent with these results, decreased accumulation of Fe and Zn was observed in flag leaves of osvit1 and osvit2 mutants. However, both mutants showed no significant change of Mn in these tissues or in grains. These results suggest that OsVIT1 and OsVIT2 may function primarily in flag leaves to mediate vacuolar sequestration of Fe and Zn. Moreover, the Fe and Zn accumulation in seeds and the decrease of those metals in flag leaves (the source organ) of osvit1 and osvit2 mutants indicates an indirect involvement of both gene products in translocation of both metals to sink organs, since they are only weakly expressed in embryos, which is in contrast to AtVIT1. Finally, there are two VIT homologs in wheat, which are located to the tonoplast (Figure 3C), but only one of them, namely TaVIT2, was shown to complement a Mn-sensitive yeast strain, pmr1. TaVIT2 was also able to transport Fe2+ and has been employed for biofortification of wheat grains with Fe.

41Members of the CAX family are metal transporters that mediate efflux of cations into the vacuole. All plant CAX transporters characterized to date appear to be able to transport Ca2+. Nevertheless, a broad metal substrate range, including Mn2+, is a common characteristic of these proteins. Based on amino acid sequences, the plant CAX family is divided into two clusters, type 1-A and 1-B. Their capability to transport Mn2+ is not related to their phylogenetic association, with AtCAX4, OsCAX1a, and VvCAX3 being members of the type 1-A subfamily and AtCAX2, AtCAX5, OsCAX3, and OsCAX4 being members of the type 1-B subfamily. AtCAX2 transports a range of cations into the vacuole, including Mn2+, Zn2+, and Cd2+. Knockout analysis suggested that AtCAX2 does not play a major physiological role in Ca homeostasis, but is more important for vacuolar Mn accumulation. However, vacuolar Mn2+ transport was not completely abolished in cax2. AtCAX5 is a likely candidate for this residual activity. AtCAX5 showed a lower Ca2+ and Mn2+ transport activity than AtCAX2 as also a reduced Ca2+ transport capacity. Despite the significant sequence similarity and substrate specificity of AtCAX2 and AtCAX5, a clear distinction appeared in their expression pattern and transcriptional regulation. AtCAX5 was expressed in all tissues of Arabidopsis seedlings, particularly in the stem and the root, and at a lower level in the leaf. In addition, there was an increase in AtCAX5 transcripts under conditions of excess Mn, and a reduction in response to excess Zn. In contrast, AtCAX2 was detected at fairly low levels in all tissues, but was not greatly induced by any metal. The relevance of these CAX transporters in intracellular Mn homeostasis is unclear. Since only the Arabidopsis cax2 mutant showed a growth defect under Mn deficiency, future genetic analysis by using cax2cax5 double mutant plants may uncover the physiological function of these transporters in plants.

42CAX2-like transporters of other species, as tomato LeCAX2 and barley HvCAX2, transport Ca2+ and Mn2+ into yeast vacuoles upon heterologous expression, but with different transport kinetics. HvCAX2 is expressed ubiquitously in roots, shoot, immature spikes, and in seeds, preferentially in the embryo rather than the endosperm. Transcripts of HvCAX2 increased under excess Ca2+ and Na+. Likewise, LeCAX2 was also expressed in roots and to a higher extend in leaves and fruits.

43AtCAX4 was expressed at low level in all tissues, and expression increased after Mn2+, Na+, and Ni2+ treatment. Specifically, AtCAX4 transcript levels increased in the root apex and lateral root primordia upon Mn2+ and Ni2+ treatment and decreased if Ca2+ was depleted. Mutation of AtCAX4 led to an arrested growth in seedlings under excess Cd2+, Mn2+, and auxin. Previously, a link between auxin-regulated plant development, cytosolic Ca2+, and kinases was described. Therefore, these findings suggest that the cax4 mutant may have increased cytosolic Ca2+ levels (because of reduced Ca2+ transport into the vacuole), that cause an impaired auxin gradient and altered root development. Alternatively, the auxin sensitivity phenotype may be a result of altered Mn homeostasis in cax4 mutants, since Mn has an influence on auxin responses and auxin metabolism. VvCAX3 is a ubiquitously expressed vacuolar transporter for both, monovalent and divalent cations in grapevine. Expression of VvCAX3 in yeast restored the growth on media with high Na+, Li+, Cu2+, and Mn2+ concentrations. Interestingly, expression of VvCAX3 decreased during the development in parallel with Ca2+ accumulation in the fruit. It is likely that VvCAX3 is not only involved in Ca2+ sequestration, but also in the detoxification of trace elements and the mitigation of salt stress, since its transcript level increased under NaCl treatment.

44Other members of the type 1-B subfamily of CAX transporters are OsCAX3 and OsCAX4 in rice. OsCAX3 is expressed in all plant tissues, and the protein has a capacity to transport Mn2+. Interestingly, when expressed in yeast, next to Mn tolerance OsCAX3 also conferred Ca2+ tolerance, but to a lower extent compared to other OsCAX transporters. Therefore, OsCAX3, as AtCAX2, might preferentially transport Mn2+, rather than Ca2+, into the vacuole. Nevertheless, future work is needed to confirm its vacuolar localization. By contrast, expression of OsCAX4 provides Mn and Cu tolerance to yeast, and OsCAX4 transcripts were increased upon prolonged salt stress in planta. A type 1-A transporter of rice, OsCAX1a, transports Ca2+ and Mn2+ into yeast vacuoles, but is involved mainly in Ca2+ homeostasis in plant cells under high concentrations of Ca2+. Further analysis of the function of OsCAX1a in planta showed that the transcript level in roots was increased by Ca2+ and decreased by Mn2+, Ni2+, Mg2+, and Cu2+, while shoots showed only an increase of OsCAX1a transcript levels after treatment with Ca2+. The decreased expression of OsCAX1a in response to other divalent cations might be a mechanism to keep high concentrations of cytosolic Ca2+ in order to protect the cell from toxic levels of ions like Mn2+. Therefore, OsCAX1a may transport Ca2+ into the vacuole under Ca2+ toxicity or regulate Ca2+ homeostasis in the cytosol. In rice, further studies using combined mutants of OsCAX1a, OsCAX3, and OsCAX4 are needed to confirm their relevance in plant Mn2+ and Ca2+ homeostasis.

45Members of the CCX family were previously described as transporters of the CAX family. However, because they are phylogenetically closer to the mammalian plasma membrane K+-dependent Na+/Ca2+ exchangers (NCXs), this gene family, which has five members in Arabidopsis (CCX1-5), was re-classified. Expression of AtCCX3 was induced in roots and flowers upon treatment with Mn2+, and in yeast the expression of AtCCX3 complemented strains defective in Mn2+ export. Tobacco cells expressing AtCCX3 showed enhanced Mn levels, further suggesting an ability of AtCCX3 to transport Mn2+. The transcript level of AtCCX3 increased in plants upon a treatment with Na+, K+, and Mn2+, albeit a mutation in AtCCX3 provoked no discernible growth abnormalities under those conditions.

46The genome of Arabidopsis contains six genes encoding NRAMP transporters, whereby AtNRAMP3 and AtNRAMP4 have been localized to the tonoplast. The NRAMP homologs TcNRAMP3 and TcNRAMP4 from Thlaspi caerulescens were also localized to the tonoplast. These NRAMP transporters from both species are involved in the transport of Cd2+, Fe2+, and Mn2+. In addition, AtNRAMP4 and TcNRAMP4 also transport Zn2+ when expressed in yeast. AtNRAMP3 and AtNRAMP4 protein levels in leaves were unaffected by Mn deficiency, but expression of AtNRAMP4 was induced under Fe-limited conditions. Both proteins were shown to be responsible for the retrieval of Fe from vacuoles during seed germination and to mediate the export of vacuolar Mn in photosynthetic tissues of adult plants. The nramp3nramp4 double mutant had comparable Mn concentrations in leaf mesophyll cells as wild type plants, but it showed a strong accumulation of Mn in the vacuoles. Under Mn deficiency, the double mutant showed a decreased growth which was correlated with reduced photosynthetic activity as a consequence of a shortage of Mn for the formation of OEC complexes in PSII. In contrast, nramp3nramp4 did not show altered mitochondrial MnSOD activity under Mn deficiency. These results suggest an important role for AtNRAMP3/AtNRAMP4-dependent Mn transit through the vacuole prior to the import into chloroplasts of mesophyll cells.

47Mn plays an extremely important role in chloroplast function, as it is essential for photosynthetic activity. There is a high demand for Mn in the photosynthetic apparatus, mainly for the formation of the Mn4Ca -cluster in the OEC of PSII, which is essential for water splitting. In spite of the importance of Mn for chloroplast function, only recent studies have reported the identification and characterization of Mn transporters in chloroplast membranes. Two members of the BICAT family, which are related to the GDT1 protein in yeast and the Mnx protein in the cyanobacterium Synechocystis, are involved in Mn loading of the chloroplast and the distribution within this organelle (Figure 3A).

48The Arabidopsis AtBICAT1 (syn. PAM71, CCHA1) protein is localized in the thylakoid membrane and transports Ca2+ and Mn2+ into the thylakoid lumen. AtBICAT1 complemented the Mn-sensitive phenotype of the pmr1 yeast mutant and mediated Ca2+ influx upon expression in E. coli. bicat1 knockout mutants showed slightly pale green leaves along with compromised growth. Interestingly, growth retardation and photosynthetic activity of bicat1 could be partially restored by exogenous treatment with Mn2+. In addition, transient elevations of the stromal free Ca2+ concentration, induced by a light-to-dark shift, were increased in bicat1 mutants. Based on these findings, AtBICAT1 presumably functions in Mn2+ and Ca2+ flux into thylakoids for assembly of the Mn4Ca-cluster, and also in the homeostasis of stromal Ca2+, which regulates numerous processes in chloroplasts.

49AtBICAT2 (syn. PAM71HL, CMT1), the second member of this family, was also described to fulfill functions in chloroplast Mn and Ca2+ homeostasis. In contrast to AtBICAT1, it resides in the chloroplast envelope. Like AtBICAT1, the expression of AtBICAT2 can also alleviate the Mn2+ and Ca2+ sensitivity phenotypes of the pmr1 and pmr1gdt1 yeast mutants, respectively. Disruption of AtBICAT2 resulted in strong chlorosis, severely impaired plant growth, defective thylakoid stacking, and severe reduction of PS II complexes, resulting in diminished photosynthetic activity. Consistent with a reduced oxygen evolution capacity, bicat2 mutant chloroplasts contained less Mn than those of the wild type. Also, mutants were defective in Ca2+ uptake across the chloroplast envelope and showed a strongly dampened dark-induced Ca2+ signal in the stroma. As consequence, the disruption of AtBICAT2 should also lead to a decreased Ca2+ import into the thylakoid lumen. Taken together, these results indicate that AtBICAT2 functions as an inner envelope transporter responsible for chloroplast Mn2+ and Ca2+ uptake. Ca2+ dynamics and Mn concentrations in both, stroma and lumen, were likely diminished in bicat2 mutants, which may explain the stronger phenotype compared to that of bicat1 mutants. The phenotype of bicat1bicat2 double mutants suggest that AtBICAT2 is the limiting step in Mn2+ and Ca2+ delivery to the chloroplast. Further work is needed to unravel the bi-functionality and regulation of BICATs in Ca2+ and Mn2+ homeostasis in chloroplasts.

50The Arabidopsis CDF protein AtMTP11 was shown to exhibit an important role in Mn detoxification. The promoter of AtMTP11 had a high activity in root tips, shoot margins, and hydathodes, but not in epidermal cells and trichomes. Expression of AtMTP11 in yeast complemented the Mn2+ hypersensitivity of the pmr1 mutant. In Arabidopsis, mtp11 mutants were hypersensitive to elevated Mn2+ levels, whereas AtMTP11-overexpressing lines were hypertolerant. As AtMTP11 appeared to be localized to the prevacuolar compartment (PVC) or to the Golgi apparatus (Figure 3A), it was suggested that it functions in the accumulation of excess Mn either into vacuoles via the PVC or in the secretion to the apoplastic space via vesicle-mediated exocytosis. The latter pathway was supported by increased Mn concentrations in mtp11 mutants. Two poplar homologs of this protein, PtMTP11.1 and PtMTP11.2, were also able to complement the Mn-sensitive yeast mutant pmr1 and targeted to the same Golgi compartments as AtMTP11 (Figure 3C; ). Those genes are therefore likely to function in a similar way. Another MTP11 ortholog in rice, OsMTP11, was also described to be involved in Mn detoxification. OsMTP11 is induced by high Mn and expressed specifically in conducting tissues. Interestingly, epigenetic mechanisms (e.g., DNA methylation) were a major factor regulating the expression level of OsMTP11. Knockdown of OsMTP11 resulted in growth inhibition in the presence of high concentrations of Mn2+ and also led to increased accumulation of Mn in shoots and roots. By contrast, the overexpression of OsMTP11 enhanced Mn tolerance of rice and decreased the accumulation of Mn in shoots and roots. Stable expression of OsMTP11-GFP in Arabidopsis and transient expression of this construct in rice and tobacco protoplasts showed that OsMTP11 was also located to the Golgi (Figure 3B; ). However, recent studies suggested that OsMTP11 localized to the trans-Golgi network (TGN) when it was expressed in rice protoplasts and tobacco epidermal cells. Surprisingly, in tobacco epidermal cells, OsMTP11 relocalized to the plasma membrane upon treatment with high extracellular Mn2+ concentrations. These findings suggest that OsMTP11 is required for Mn homeostasis and contributes to Mn2+ tolerance in rice.

51In the sugar beet relative B. vulgaris spp. maritima, the MTP11 homologs BmMTP10 and BmMTP11 were also characterized as Mn2+ transporters by complementation of the pmr1 yeast strain. In barley, transient expression in onion epidermal cells showed that the HvMTP8.1 and HvMTP8.2 proteins, which are most closely related to the vacuolar AtMTP8 (see section Vacuoles as Manganese Stores), were localized to the Golgi apparatus and also complemented the pmr1 strain. However, the function of those CDF proteins in planta is still unclear.

52The Arabidopsis genome encodes 15 P-type Ca2+-ATPases, of which the P2A -type or ECA (ER-type Calcium ATPase) subfamily has two members involved in endomembrane Mn2+ transport. AtECA1 and AtECA3 both function as a pump for Ca2+ and Mn2+, and localize to the ER and Golgi apparatus, respectively (Figure 3A). Furthermore, AtECA3 was shown to be localized also in subpopulations of endosomes or PVC. AtECA1 and AtECA3 expression in the K616 yeast mutant, defective in the Golgi Ca2+ and Mn2+ pump PMR1 and in the vacuolar Ca2+ pump PMC1, increased its tolerance to toxic levels of Mn2+ and to Ca2+ deficiency. Another ER-localized ECA transporter in tomato, LCA1, also complemented the growth of K616 yeast under conditions of high Mn2+ and low Ca2+. eca1 mutants of Arabidopsis showed impaired growth under Ca2+ deprivation and Mn2+ excess. On high-Mn2+ media, root hair elongation was inhibited in the mutants, suggesting an impairment in tip growth. Taken together, AtECA1 and LeLCA1 are likely involved in the transport of Ca2+ and Mn2+ from the cytosol into the ER. On the other hand, eca3 was shown to be sensitive to Mn deficiency and also to Mn toxicity, but not to Ca2+ deficiency. Thus, the phenotype of eca3, observed under Mn-deficient conditions, may be due to a reduction of the Mn content in the Golgi. Based on these studies, CDF and ECA proteins may be responsible for Mn2+ loading of Golgi-related vesicular compartments, for delivering Mn to Mn-dependent enzymes and/or for detoxification of Mn via a secretory pathway.

53In Arabidopsis, AtNRAMP2 is located in the TGN (Figure 3A) and was shown to be involved in the intracellular allocation of Mn. AtNRAMP2 is mainly expressed in the vasculature of roots and shoots and was barely induced upon Mn deficiency. Nevertheless, nramp2 mutants showed a hypersensitivity to Mn deficiency and a reduction in Mn contents in vacuoles and chloroplasts with an accompanying reduction in PSII activity under those conditions. Surprisingly, the nramp2nramp3nramp4 triple mutant did not exhibit higher sensitivity to Mn deficiency than the single nramp2 and double nramp3nramp4 mutants. This suggests that the three transporters act in the same pathway to deliver Mn to the chloroplasts.

54NRAMP transporters from tomato (LeNRAMP1 and LeNRAMP3) and apple (MbNRAMP1) were also able to transport Mn2+ and located to intracellular vesicles when expressed in yeast cells, but their function in Mn homeostasis is still unclear.

55ER bodies are fusiform compartments connected to the ER that were found specifically in Brassicales. It has been proposed that ER bodies might play a role in the defense against pathogens and herbivores. In Arabidopsis, two VIT transporters, AtMEB1 (Membrane protein of Endoplasmic reticulum Body) and AtMEB2, that localize to the ER body membrane but not to the ER network, have been identified. Heterologous expression of AtMEB1 and AtMEB2 in yeast suppressed Fe and Mn toxicity, suggesting that they possibly act as metal transporters. ER bodies are present in hypocotyls of seedlings where they disappear during plant development. In contrast, in roots, ER bodies are constitutively present. Therefore, it has been suggested that MEB transporters may be involved in the sequestration of metals into root ER bodies under metal stress conditions.

56In mitochondria, Mn is crucial for the activity of MnSOD which scavenges ROS generated within the citric acid cycle and the electron transport chain. However, the transport mechanisms for Mn2+ in plant mitochondria still remain to be elucidated. To date, no transporter involved in Mn2+ transport to and from plant mitochondria has been characterized. Nevertheless, in humans, a mitochondrial Ca2+ uniporter (MCU) that is responsible for Ca2+ and Mn2+ loading into the mitochondria has been described. Arabidopsis has six MCU isoforms which are predicted to be localized in mitochondria, except one, cMCU, that is targeted to the chloroplast. The cMCU protein mediates Ca2+ fluxes across the chloroplast envelope. In humans, MCU is part of a complex named MCUC (MCU complex) which includes other subunits, including the EF hand-containing proteins MICU1 and MICU2 (Ca2+-sensing inhibitory subunit). MICU1 plays a decisive role in ion specificity of MCU, allowing it to distinguish between Ca2+ vs. Mn2+. In Arabidopsis, a MICU homolog, AtMICU, that binds Ca2+ and localizes to the mitochondria, was described. These findings provoke the idea that a conserved uniporter system, similar to MCUC in humans, may mediate Ca2+ and Mn2+ uptake by plant mitochondria.

57In peroxisomes, Mn is important as a cofactor of the peroxisomal MnSOD. This enzyme is present in some plant species, including pea, cucumber, and pepper. However, so far, transporters to load Mn into the peroxisomes are still missing.

58The relevance of manganese as a micronutrient of plants is still largely underestimated. For a long time, the accepted dogma among animal and plant biologists has been that the physiological requirement for Mn by living cells is low and that Mn uptake exceeds the requirement. However, in natural and in agricultural settings, Mn availability can be a seriously limiting factor for plant growth, which necessitates the operation of high-affinity Mn transporters in roots and efficient mechanisms of Mn distribution in the plant to cope with Mn shortage. Crops with improved Mn uptake capacity and Mn use efficiency will achieve higher growth and yield under suboptimal Mn availability, primarily by providing sufficient Mn to PSII and thus increasing their photosynthetic efficiency.

59There are many unresolved issues in our understanding of Mn transport and homeostasis, such as the transport of Mn into the xylem – Is it mediated by a vesicle-based secretory mechanism or by plasma membrane-bound exporters? How Mn is imported by different cell types in the shoot is another open question. Most Mn2+ transporters are not specific, but also able to move other divalent cations, like Ca2+, Fe2+, Zn2+, or Cu2+, but so far it is still unclear if and how they discriminate between the cations. In humans, the substrate selectivity of a mitochondrial Mn transporter is altered by interacting EF hand proteins. In this respect, the ability of diverse Ca2+ transport proteins to also permeate Mn2+ (or vice versa) is particular interesting. The possibility that Ca2+ and Mn2+ share transport pathways mediated by ECAs, BICATs, CAXs, and Ca2+ channels, implies an interference of Mn2+ in Ca2+ signaling. In fact, Mn2+ has been demonstrated to act as a signaling agent per se in humans.

60The sensing and signaling of the plant’s Mn status is another area yet to be explored: On which level is Mn transport activity regulated by the nutritional status of the plant? This review pointed out some transcriptional changes of Mn transporter-encoding genes under Mn-toxic and Mn-deficient conditions, but the signaling networks that underlie these changes are still missing. Furthermore, a recent systems-wide analysis indicated that Mn deficiency responses are primarily regulated at the posttranscriptional level. Moreover, our understanding of how Mn transport proteins are regulated is very rudimentary. In this respect, a couple of proteins that regulate the subcellular localization of AtNRAMP1 have been identified, revealing links to inositol phosphate and choline homeostasis. As another fascinating case, it has been demonstrated that phosphorylation, ubiquitination, and metal binding modify the stability and localization of AtIRT1, an Fe2+ transporter that contributes to the uptake of Mn2+. However, for the vast majority of Mn transporters, posttranslational modifications and protein-protein interactions are unknown.

61Future research has to be focused on all these fundamental aspects. The function, regulation, and co-operation of Mn2+ transport proteins in different plant tissues upon Mn deficiency and excess ought to be understood at transcriptional and at protein level. Such studies will eventually elucidate the mechanisms controlling Mn acquisition, subcellular compartmentation, and homeostasis in plants, a knowledge that can be harnessed to develop Mn-efficient germplasm of staple crops.

62SA, SH, and BM drafted the manuscript. SA, SH, BM, and EP finalized the manuscript, approved the final version of the manuscript, and agreed to be accountable for all aspects of the work.

63The 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.