Manganese Tolerance Research Articles

A member of cation diffusion facilitator family, MTP11, is required for manganese tolerance and high fertility in rice.

Rice MTP11 is the trans-Golgi-localized transporter that is involved in Mn tolerance with MTP8.1, and it is required for normal fertility. Rice (Oryza sativa L.) is one of the most manganese (Mn)-tolerant species, and it is able to accumulate high levels of this metal in the leaves without showing toxic symptoms. The metal tolerance protein 8.1 (MTP8.1), a member of the Mn-cation diffusion facilitator (CDF) family, has been shown to play a central role in high Mn tolerance by sequestering Mn into vacuoles. Recently, rice MTP11 was identified as an Mn transporter that is localized to Golgi-associated compartments, but its exact role in Mn tolerance in planta has not yet been understood. Here, we investigated the role of MTP11 in rice Mn tolerance using knockout lines. Old leaves presented higher levels of constitutively expressed MTP11 than other tissues and MTP11 expression was also found in reproductive organs. Fused MTP11:green fluorescent protein was co-localized to trans-Golgi markers and differentiated from other Golgi-associated markers. Knockout of MTP11 in wild-type rice did not affect tolerance and accumulation of Mn and other heavy metals, but knockout in the mtp8.1 mutant showed exacerbated Mn sensitivity at the vegetative growth stage. Knockout of MTP11 alone resulted in decreased grain yield and fertility at the reproductive stage. Thus, MTP11 is a trans-Golgi localized transporter for Mn, which plays a role in Mn tolerance through intracellular Mn compartmentalization. It is also required for maintaining high fertility in rice.

High Manganese Tolerance and Biooxidation Ability of Serratia marcescens Isolated from Manganese Mine Water in Minas Gerais, Brazil.

Manganese is an important metal for the maintenance of several biological functions, but it can be toxic in high concentrations. One of the main forms of human exposure to metals, such as manganese (Mn), is the consumption of solar salt contaminated. Mn-tolerant bacteria could be used to decrease the concentration of this metal from contaminated sites through safer environmental-friendly alternative technology in the future. Therefore, this study was undertaken to isolate and identify Mn resistant bacteria from water samples collected from a Mn mine in the Iron Quadrangle region (Minas Gerais, Brazil). Two bacterial isolates were identified as Serratia marcescens based on morphological, biochemical, 16S rDNA gene sequencing and phylogeny analysis. Maximum resistance of the selected isolates against increasing concentrations of Mn(II), up to 1200 mg L-1 was determined in solid media. A batch assay was developed to analyze and quantify the Mn removal capacities of the isolates. Biological Mn removal capacities of over 55% were detected for both isolates. Whereas that mechanism like biosorption, precipitation and oxidation could be explaining the Mn removal, we seek to give an insight into some of the molecular mechanisms adopted by S. marcescens isolates. For this purpose, the following approaches were adopted: leucoberbelin blue I assay, Mn(II) oxidation by cell-free filtrate and electron microscopy and energy-dispersive X-ray spectroscopy analyses. Overall, these results indicate that S. marcescens promotes Mn removal in an indirect mechanism by the formation of Mn oxides precipitates around the cells, which should be further explored for potential biotechnological applications for water recycling both in hydrometallurgical and mineral processing operations.

The transporter SynPAM71 is located in the plasma membrane and thylakoids, and mediates manganese tolerance in Synechocystis PCC6803.

Manganese (Mn) is an essential constituent of photosystem II (PSII) and therefore indispensable for oxygenic photosynthesis. Very little is known about how Mn is transported, delivered and retained in photosynthetic cells. Recently, the thylakoid-localized transporter PAM71 has been linked to chloroplast Mn homeostasis in Arabidopsis thaliana. Here, we characterize the function of its homolog in Synechocystis (SynPAM71). We used a loss-of-function line (ΔSynPAM71), wild-type (WT) cells exposed to Mn stress and strains expressing a tagged variant of SynPAM71 to characterize the role of SynPAM71 in cyanobacterial Mn homeostasis. The ΔSynPAM71 strain displays an Mn-sensitive phenotype with reduced levels of chlorophyll and PSI accumulation, defects in PSII photochemistry and intracellular Mn enrichment, particularly in the thylakoid membranes. These effects are attributable to Mn toxicity, as very similar symptoms were observed in WT cells exposed to excess Mn. Moreover, CyanoP, which is involved in the early steps of PSII assembly, is massively upregulated in ΔSynPAM71. SynPAM71 was detected in both the plasma membrane and, to a lesser extent, the thylakoid membranes. Our results suggest that SynPAM71 is involved in the maintenance of Mn homeostasis through the export of Mn from the cytoplasm into the periplasmic and luminal compartments, where it can be stored without interfering with cytoplasmic metabolic processes.

Metal transport protein 8 in Camellia sinensis confers superior manganese tolerance when expressed in yeast and Arabidopsis thaliana

Manganese (Mn) is an important micronutrient element required for plant growth and development, playing catalytic roles in enzymes, membranes and DNA replication. The tea plant (Camellia sinensis) is able to accumulate high concentration of Mn without showing signs of toxicity, but the molecular mechanisms underlying this remain largely unknown. In this study, the C. sinensis cultivar ‘LJCY’ had higher Mn tolerance than cultivar ‘YS’, because chlorophyll content reduction was lower under the high Mn treatment. Proteomic analysis of the leaves revealed that C. sinensis Metal Tolerance Protein 8 (CsMTP8) accumulated in response to Mn toxicity in cultivar ‘LJCY’. The gene encoding CsMTP8, designated as CsMTP8 was also isolated, and its expression enhanced Mn tolerance in Saccharomyces cerevisiae. Similarly, the overexpression of CsMTP8 in Arabidopsis thaliana increased plant tolerance and reduced Mn accumulation in plant tissues under excess Mn conditions. Subcellular localization analysis of green florescence fused protein indicated that CsMTP8 was localized to the plasma membranes. Taken together, the results suggest that CsMTP8 is a Mn-specific transporter, which is localized in the plasma membrane, and transports excess Mn out of plant cells. The results also suggest that it is needed for Mn tolerance in shoots.

Identification of manganese-toxicity-responsive genes in roots of two citrus species differing in manganese tolerance using cDNA-AFLP

We identified more Mn-toxicity-responsive genes from Mn-intolerant Citrus grandis than from Mn-tolerant Citrus sinensis roots. These findings increased our understanding of the molecular mechanisms on plant Mn toxicity and Mn tolerance. Manganese (Mn) toxicity is the most important factor limiting crop production after aluminum toxicity in acidic soils. However, little is known about Mn-toxicity-induced alterations of gene expression profiles in woody plants. Using cDNA-AFLP, we identified 87 and 63 Mn-toxicity-responsive genes from Mn-intolerant ‘Sour pummelo’ (Citrus grandis) and Mn-tolerant ‘Xuegan’ (Citrus sinensis) roots. Among these genes, only 22 genes with the same accession number were shared by both. Protein phosphorylation/dephosphorylation-related genes were upregulated in C. sinensis roots, and downregulated in C. grandis roots except for one differentially expressed gene. Sulfur metabolism-related genes were repressed only in Mn-toxic C. grandis roots. Obviously, great differences existed in Mn-toxicity-induced alterations of gene expression profiles between C. sinensis and C. grandis roots. Genes related to protein phosphorylation/dephosphorylation (i.e., cyclin-dependent kinase-activating kinase assembly factor-related protein and PP2A regulatory subunit TAP46), cellular transport (i.e., Ca-transporting ATPase 1), and nucleic acid (i.e., ethylene-responsive transcription factor ERF109-like, structural maintenance of chromosomes protein 4-like, RNA-binding protein and DEAD-box ATP-dependent RNA helicase 21), cell wall (i.e., pectin methylesterase 1 and invertase/pectin methylesterase inhibitor family protein) and fatty acid (i.e., carboxylesterase 20) metabolisms might play a role in C. sinensis Mn tolerance. In addition, cell wall materials were increased in Mn-toxic C. sinensis and C. grandis roots, especially in the former. Interestingly, lignin content was increased in Mn-toxic C. sinensis roots, while a reverse trend was displayed in Mn-toxic C. grandis roots. In conclusion, our results provided novel clues to the molecular mechanisms on Mn toxicity and Mn tolerance in higher plants.

Manganese-II oxidation and Copper-II resistance in endospore forming Firmicutes isolated from uncontaminated environmental sites

The accumulation of metals in natural environments is a growing concern of modern societies since they constitute persistent, non-degradable contaminants. Microorganisms are involved in redox processes and participate to the biogeochemical cycling of metals. Some endospore-forming Firmicutes (EFF) are known to oxidize and reduce specific metals and have been isolated from metal-contaminated sites. However, whether EFF isolated from uncontaminated sites have the same capabilities has not been thoroughly studied. In this study, we measured manganese oxidation and copper resistance of aerobic EFF from uncontaminated sites. For the purposes of this study we have sampled 22 natural habitats and isolated 109 EFF strains. Manganese oxidation and copper resistance were evaluated by growth tests as well as by molecular biology. Overall, manganese oxidation and tolerance to over 2 mM copper was widespread among the isolates (more than 44% of the isolates exhibited Mn (II)-oxidizing activity through visible Birnessite formation and 9.1% tolerate over 2 mM copper). The co-occurrence of these properties in the isolates was also studied. Manganese oxidation and tolerance to copper were not consistently found among phylogenetically related isolates. Additional analysis correlating the physicochemical parameters measured on the sampling sites and the metabolic capabilities of the isolates showed a positive correlation between in situ alkaline conditions and the ability of the strains to perform manganese oxidation. Likewise, a negative correlation between temperature in the habitat and copper tolerance of the strains was observed. Our results lead to the conclusion that metal tolerance is a wide spread phenomenon in unrelated aerobic EFF from natural uncontaminated environments.

Manganese tolerance in yeasts involves polyphosphate, magnesium, and vacuolar alterations.

Basidiomycetous and ascomycetous yeast species were tested for manganese tolerance. Basidiomycetous Cryptococcus humicola, Cryptococcus terricola, Cryptococcus curvatus and ascomycetous Candida maltosa, Kluyveromyces marxianus, Kuraishia capsulata, Lindnera fabianii and Sacharomyces cerevisiae were able to grow at manganese excess (2.5mmol/L), while the growth of basidiomycetous Rhodotorula bogoriensis was completely suppressed. The lag phase duration increased and the exponential growth rate decreased at manganese excess. The increase of cell size and enlargement of vacuoles were characteristics for the cells grown at manganese excess. The alterations in inorganic polyphosphate content and cellular localization were studied. L. fabianii, K. capsulata, C. maltosa, and Cr. humicola accumulated the higher amounts of inorganic polyphosphates, while Cr. terricola and Cr. curvatus demonstrated no such accumulation. The polyphosphate content in the cell wall tested by DAPI staining increased in all species under the study; however, this effect was more pronounced in Cr. terricola and Cr. curvatus. The accumulation of Mg(2+) in the cell wall under Mn(2+) excess was observed in Cr. humicola, Cr. curvatus and Cr. terricola. The accumulation of polyphosphate and magnesium in the cell wall was supposed to be a factor of manganese tolerance in yeasts.

Malate synthesis and secretion mediated by a manganese-enhanced malate dehydrogenase confers superior manganese tolerance in Stylosanthes guianensis.

Manganese (Mn) toxicity is a major constraint limiting plant growth on acidic soils. Superior Mn tolerance in Stylosanthes spp. has been well documented, but its molecular mechanisms remain largely unknown. In this study, superior Mn tolerance in Stylosanthes guianensis was confirmed, as reflected by a high Mn toxicity threshold. Furthermore, genetic variation of Mn tolerance was evaluated using two S. guianensis genotypes, which revealed that the Fine-stem genotype had higher Mn tolerance than the TPRC2001-1 genotype, as exhibited through less reduction in dry weight under excess Mn, and accompanied by lower internal Mn concentrations. Interestingly, Mn-stimulated increases in malate concentrations and exudation rates were observed only in the Fine-stem genotype. Proteomic analysis of Fine-stem roots revealed that S. guianensis Malate Dehydrogenase1 (SgMDH1) accumulated in response to Mn toxicity. Western-blot and quantitative PCR analyses showed that Mn toxicity resulted in increased SgMDH1 accumulation only in Fine-stem roots, but not in TPRC2001-1. The function of SgMDH1-mediated malate synthesis was verified through in vitro biochemical analysis of SgMDH1 activities against oxaloacetate, as well as in vivo increased malate concentrations in yeast (Saccharomyces cerevisiae), soybean (Glycine max) hairy roots, and Arabidopsis (Arabidopsis thaliana) with SgMDH1 overexpression. Furthermore, SgMDH1 overexpression conferred Mn tolerance in Arabidopsis, which was accompanied by increased malate exudation and reduced plant Mn concentrations, suggesting that secreted malate could alleviate Mn toxicity in plants. Taken together, we conclude that the superior Mn tolerance of S. guianensis is achieved by coordination of internal and external Mn detoxification through malate synthesis and exudation, which is regulated by SgMDH1 at both transcription and protein levels.

Comparison of manganese tolerance and accumulation among 24 Salix clones in a hydroponic experiment: Application for phytoremediation

Willows (Salix spp.) are excellent candidates for phytoremediation owing to their large biomass, high metal tolerance and accumulation capacity. In this study, manganese tolerance and accumulation capability in 24 Salix clones were evaluated exposed to 1mM Mn by a hydroponic system for 21days. Results suggested that there were wide variations in Mn tolerance and accumulation capability among the clones. Clonal variation in biomass production ranged from growth reduction to growth stimulation. The clonal differences in Mn concentrations (μgg−1, dry weight, DW) ranged from 3183.10 to 5827.7 in leaves, from 1840.48 to 4572.17 in stems, and from 2733.33 to 10,253.88 in roots exposed to excess Mn. The total Mn contents in shoots (including leaves and stems) varied 5.8-fold among clones under Mn treatment. Five clones exhibited high Mn tolerance and accumulation capacity, and clone J333 (Salix babylonica×Salix matsudana) had a relatively high Mn tolerance index and the highest Mn content in aboveground tissues. Consequently, further evaluation of the Salix clones for Mn tolerance and phytoremediation potential is recommended in field experiments.

Leaf cDNA-AFLP analysis of two citrus species differing in manganese tolerance in response to long-term manganese-toxicity

BackgroundVery little is known about manganese (Mn)-toxicity-responsive genes in citrus plants. Seedlings of ‘Xuegan’ (Citrus sinensis) and ‘Sour pummelo’ (Citrus grandis) were irrigated for 17 weeks with nutrient solution containing 2 μM (control) or 600 μM (Mn-toxicity) MnSO4. The objectives of this study were to understand the mechanisms of citrus Mn-tolerance and to identify differentially expressed genes, which might be involved in Mn-tolerance.ResultsUnder Mn-toxicity, the majority of Mn in seedlings was retained in the roots; C. sinensis seedlings accumulated more Mn in roots and less Mn in shoots (leaves) than C. grandis ones and Mn concentration was lower in Mn-toxicity C. sinensis leaves compared to Mn-toxicity C. grandis ones. Mn-toxicity affected C. grandis seedling growth, leaf CO2 assimilation, total soluble concentration, phosphorus (P) and magenisum (Mg) more than C. sinensis. Using cDNA-AFLP, we isolated 42 up-regulated and 80 down-regulated genes in Mn-toxicity C. grandis leaves. They were grouped into the following functional categories: biological regulation and signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, lipid metabolism, cell wall metabolism, stress responses and cell transport. However, only 7 up-regulated and 8 down-regulated genes were identified in Mn-toxicity C. sinensis ones. The responses of C. grandis leaves to Mn-toxicity might include following several aspects: (1) accelerating leaf senescence; (2) activating the metabolic pathway related to ATPase synthesis and reducing power production; (3) decreasing cell transport; (4) inhibiting protein and nucleic acid metabolisms; (5) impairing the formation of cell wall; and (6) triggering multiple signal transduction pathways. We also identified many new Mn-toxicity-responsive genes involved in biological and signal transduction, carbohydrate and protein metabolisms, stress responses and cell transport.ConclusionsOur results demonstrated that C. sinensis was more tolerant to Mn-toxicity than C. grandis, and that Mn-toxicity affected gene expression far less in C. sinensis leaves. This might be associated with more Mn accumulation in roots and less Mn accumulation in leaves of Mn-toxicity C. sinensis seedlings than those of C. grandis seedlings. Our findings increase our understanding of the molecular mechanisms involved in the responses of plants to Mn-toxicity.

Characterization of two genes encoding metal tolerance proteins from Beta vulgaris subspecies maritima that confers manganese tolerance in yeast

Manganese (Mn(2+)) is an essential micronutrient in plants. However increased Mn(2+) levels are toxic to plant cells. Metal tolerance proteins (MTPs), member of cation diffusion facilitator protein (CDF) family, have important roles in metal homeostatis in different plant species and catalyse efflux of excess metal ions. In this study, we identified and characterized two MTP genes from Beta vulgaris spp. maritima (B. v. ssp. maritima). Overexpression of these two genes provided Mn tolerance in yeast cells. Sequence analyses displayed BmMTP10 and BmMTP11 as members of the Mn-CDF family. Functional analyses of these proteins indicated that they are specific to Mn(2+) with a role in reducing excess cellular Mn(2+) levels when expressed in yeast. GFP-fusion constructs of both proteins localized to the Golgi apparatus as a punctuated pattern. Finally, Q-RT-PCR results showed that BmMTP10 expression was induced threefold in response to the excess Mn(2+) treatment. On the other hand BmMTP11 expression was not affected in response to excess Mn(2+) levels. Thus, our results suggest that the BmMTP10 and BmMTP11 proteins from B. v. ssp. maritima have non-redundant functions in terms of Mn(2+) detoxification with a similar in planta localization and function as the Arabidopsis Mn-CDF homolog AtMTP11 and this conservation shows the evolutionary importance of these vesicular proteins in heavy metal homeostatis among plant species.

Investigation of manganese tolerance and accumulation of two Mn hyperaccumulators Phytolacca americana L. and Polygonum hydropiper L. in the real Mn-contaminated soils near a manganese mine

Up to date, very few studies have examined the phytoextraction ability of hyperaccumulators in the real soils contaminated with heavy metals following the identification of the hyperaccumulators using hydroponics tests. In the present study, amended with a chemical-mobilizing agent of ethylene diamine triacetic acid (EDTA), the manganese accumulation, tolerance and the hyperaccumulating ability of two hyperaccumulators Phytolacca americana L. (Phytolaccaceae) and Polygonum hydropiper L. (Polygonaceae) proved by previous hydroponics tests were examined using pot experiments with paddy soils contaminated with Mn (1,047 ± 53 mg kg−1) collected from one site in the vicinity of one manganese mine in Xiushan county of Chongqing, China. Results showed that the root and shoot biomass of P. americana and the chlorophyll production of P. hydropiper were significantly inhibited by EDTA treatments while the chlorophyll production of P. americana was oppositely obviously promoted by EDTA. It is noteworthy that, with or without EDTA treatment, the shoot Mn concentrations of both plant species were significantly below the hyperaccumulator threshold, indicating these two plants concealed their hyperaccumulating ability in the studied soils. Thus, further detailed studies need to be conducted to promote the metal hyperaccumulating capacity of these two plant species under comparably low Mn-contaminated soil conditions as shown in this study before the successful application of these Mn hyperaccumulators identified in laboratories to the phytoextraction of lightly or moderately Mn-contaminated agricultural soils.

The Role of the Parkinson's Disease Gene PARK9 in Essential Cellular Pathways and the Manganese Homeostasis Network in Yeast

YPK9 (Yeast PARK9; also known as YOR291W) is a non-essential yeast gene predicted by sequence to encode a transmembrane P-type transport ATPase. However, its substrate specificity is unknown. Mutations in the human homolog of YPK9, ATP13A2/PARK9, have been linked to genetic forms of early onset parkinsonism. We previously described a strong genetic interaction between Ypk9 and another Parkinson's disease (PD) protein α-synuclein in multiple model systems, and a role for Ypk9 in manganese detoxification in yeast. In humans, environmental exposure to toxic levels of manganese causes a syndrome similar to PD and is thus an environmental risk factor for the disease. How manganese contributes to neurodegeneration is poorly understood. Here we describe multiple genome-wide screens in yeast aimed at defining the cellular function of Ypk9 and the mechanisms by which it protects cells from manganese toxicity. In physiological conditions, we found that Ypk9 genetically interacts with essential genes involved in cellular trafficking and the cell cycle. Deletion of Ypk9 sensitizes yeast cells to exposure to excess manganese. Using a library of non-essential gene deletions, we screened for additional genes involved in tolerance to excess manganese exposure, discovering several novel pathways involved in manganese homeostasis. We defined the dependence of the deletion strain phenotypes in the presence of manganese on Ypk9, and found that Ypk9 deletion modifies the manganese tolerance of only a subset of strains. These results confirm a role for Ypk9 in manganese homeostasis and illuminates cellular pathways and biological processes in which Ypk9 likely functions.

Functional associations between the metabolome and manganese tolerance in Vigna unguiculata

Genotypic- and silicon (Si)-mediated differences in manganese (Mn) tolerance of cowpea (Vigna unguiculata) arise from a combination of symplastic and apoplastic traits. A detailed metabolomic inspection could help to identify functional associations between genotype- and Si-mediated Mn tolerance and metabolism. Two cowpea genotypes differing in Mn tolerance (TVu 91, Mn sensitive; TVu 1987, Mn tolerant) were subjected to differential Mn and Si treatments. Gas chromatography–mass spectrometry (GC-MS)-based metabolite profiling of leaf material was performed. Detailed evaluation of the response of metabolites was combined with gene expression and physiological analyses. After 2 d of 50 μM Mn supply TVu 91 expressed toxicity symptoms first in the form of brown spots on the second oldest trifoliate leaves. Silicon treatment suppressed symptom development in TVu 91. Despite higher concentrations of Mn in leaves of TVu 1987 compared with TVu 91, the tolerant genotype did not show symptoms. From sample cluster formation as identified by independent component analysis (ICA) of metabolite profiles it is concluded that genotypic differences accounted for the highest impact on variation in metabolite pools, followed by Mn and Si treatments in one of two experiments. Analysis of individual metabolites corroborated a comparable minor role for Mn and Si treatments in the modulation of individual metabolites. Mapping individual metabolites differing significantly between genotypes onto biosynthetic pathways and gene expression studies on the corresponding pathways suggest that genotypic Mn tolerance is a consequence of differences (i) in the apoplastic binding capacity; (ii) in the capability to maintain a high antioxidative state; and (iii) in the activity of shikimate and phenylpropanoid metabolism.

Roles of apoplastic peroxidases, laccases, and lignification in the manganese tolerance of hyperaccumulator Phytolacca americana

We investigated the response of Mn-hyperaccumulator Phytolacca americana L. to manganese excess as well as the relationships between lignin deposition in the plant’s leaves, peroxidase and laccase activities in the leaf apoplast, and Mn toxicity. The exceptionally high tolerance of P. americana to Mn, both in solution and in tissue, was confirmed. No visible brown spot was observed in the leaves of plants treated with ≤10,000 μM Mn for 10 days. Mn treatment significantly increased lignin content and laccase activity in the apoplastic washing fluid (AWF) of P. americana leaves. In contrast, an increase in the Mn supply was paralleled by a significant decrease in the concentration of total phenolic compounds (TPCs) and in water-soluble guaiacol peroxidase (SPOD) activity in leaf AWF. This result suggested that an increase in lignin deposition decreased the concentration of apoplastic TPCs that are available to generate potentially toxic intermediates by acting as peroxidase substrates. Thus, data of the present study indicate that lignin formation by laccase activities reduces Mn toxicity and increases Mn tolerance of P. americana by depressing SPOD-mediated formation of toxic intermediates from TPCs.

Manganese tolerance and accumulation in six Mn hyperaccumulators or accumulators

This study used hydroponics cultivation to investigate the manganese (Mn) accumulation and tolerance abilities of six species—Phytolacca americana L., Poa annua L., Comnyza canadensis L., Cynodon dactylon L., Polygonum hydropiper L., and Polygonum perfoliatum L. We found that P. perfoliatum, P. hydropiper, and P. americana were Mn-hyperaccumulators and that P. perforliatum have superior Mn accumulation and toleration abilities over the other five species. The Mn concentration within the shoots of P. perfoliatum reached as high as 18,342.3 mg kg−1. The root growth of P. perfoliatum was promoted under low-Mn treatments, but the growths of the five other species were inhibited by the Mn treatments and the damage intensified as Mn concentration increased. The biomass of P. perfoliatum was minimally affected by the Mn treatments. The chlorophyll (CHL), soluble protein (SP), and malondialdehyde (MDA) contents of P. perfoliatum were not adversely affected, but these parameters of the other five species showed significant (P < 0.05) deterioration from the control. By comparison among the six species, the hyperaccumulator P. perfoliatum was the most suitable species for bioremediation of Mn-polluted environments. However, the findings need further study in soil cultivation.

Safety and efficacy of Mintrex®Mn (Manganese chelate of hydroxy analogue of methionine) as feed additive for all species - Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed

Following a request from the European Commission, the European Food Safety Authority was asked to deliver a scientific opinion on the safety and efficacy of a manganese chelate of the hydroxy analogue of methionine (MintrexMn) as a feed additive for all species. MintrexMn contains a minimum of 16 % manganese and 76 % hydroxy methionine analogue ((2-hydroxy-4-methylthio)butanoic acid, HMTBa), as shown from analyses of the product. It is intended to be used as a source of the essential trace element manganese. Manganese (in several forms) and HMTBa are already separately authorised as nutritional feed additives in the European Union. In a study on chickens for fattening using a wide dose range, MintrexMn supplementation increased tibia bone deposition, an indicator of manganese bioavailability. The concentration of manganese in tibia bone supplied by MintrexMn did not differ from that supplied by manganese sulphate, a recognised source of available manganese in animal nutrition. Thus MintrexMn can be considered as a bioavailable manganese source, comparable to other authorised sources of manganese. No specific studies on tolerance in target animals to MintrexMn were provided. The efficacy study, which included supplemented doses of up to 800 mg manganese kg diet, provided some evidence of safety but alone was not adequate to draw conclusions on the safety for target animals. However, considering the published data on manganese tolerance in poultry with other manganese sources, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concludes that manganese from MintrexMn is safe for chickens for fattening.

Coffee (Coffea arabica L.) plants' tolerance to manganese in a nutrient solution.

Manganese toxicity is one of the most common toxicities among micronutrients, especially in tropical acid soils, affecting crop growth and productivity. The objective of this work was to determine differential tolerance to manganese doses in coffee plants ( Coffea arabica L.). Thus, seedlings of 14 coffee plant progenies were subjected to 7,0 and 70,0 μ mol L-1 of manganese, for seven months. The experiment was carried out in greenhouse using plastic pots containing aerated and modified Clark solution. A 14 x 2 factorial scheme (14 progenies and two manganese concentrations) arranged in a completely randomized design with three replications was used. At the end of the assay, the growth characteristics of the plants were evaluated. Manganese content was determined in the different leaf dry matter fractions (youngest leaves, oldest leaves and completely expanded leaves). When cultivated with a 70,0 μ mol L-1 manganese concentration, the progenies UFV 3880, Mundo Novo 12 CAS, H-518-3-6-1 and H-518-2-10-14 experienced a reduction in growth characteristics, suggesting high sensibility to manganese toxicity, while the progenies UFV 6869 and H-337-13- 3-10 did not present any reduction of the growth characteristics evaluated, demonstrating a high tolerance to manganese toxicity. Similarly, manganese contents in mature leaves were toxic to some progenies, but not to others, demonstrating differences among coffee varietals.

A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance

Manganese toxicity is a major problem for plant growth in acidic soils, but cellular mechanisms that facilitate growth in such conditions have not been clearly delineated. Established mechanisms that counter metal toxicity in plants involve chelation and cytoplasmic export of the metal across the plasma or vacuolar membranes out of the cell or sequestered into a large organelle, respectively. We report here that expression of the Arabidopsis and poplar MTP11 cation diffusion facilitators in a manganese-hypersensitive yeast mutant restores manganese tolerance to wild-type levels. Microsomes from yeast expressing AtMTP11 exhibit enhanced manganese uptake. In accord with a presumed function of MTP11 in manganese tolerance, Arabidopsis mtp11 mutants are hypersensitive to elevated levels of manganese, whereas plants overexpressing MTP11 are hypertolerant. In contrast, sensitivity to manganese deficiency is slightly decreased in mutants and increased in overexpressing lines. Promoter-GUS studies showed that AtMTP11 is most highly expressed in root tips, shoot margins, and hydathodes, but not in epidermal cells and trichomes, which are generally associated with manganese accumulation. Surprisingly, imaging of MTP11-EYFP fusions demonstrated that MTP11 localizes neither to the plasma membrane nor to the vacuole, but to a punctate endomembrane compartment that largely coincides with the distribution of the trans-Golgi marker sialyl transferase. Golgi-based manganese accumulation might therefore result in manganese tolerance through vesicular trafficking and exocytosis. In accord with this proposal, Arabidopsis mtp11 mutants exhibit enhanced manganese concentrations in shoots and roots. We propose that Golgi-mediated exocytosis comprises a conserved mechanism for heavy metal tolerance in plants.

The Role of Hydrogen Peroxide-Producing and Hydrogen Peroxide-Consuming Peroxidases in the Leaf Apoplast of Cowpea in Manganese Tolerance

The apoplast is considered the leaf compartment decisive for manganese (Mn) toxicity and tolerance in cowpea (Vigna unguiculata). Particularly apoplastic peroxidases (PODs) were proposed to be key enzymes in Mn toxicity-induced processes. The presented work focuses on the characterization of the role of hydrogen peroxide (H2O2)-producing (NADH peroxidase) and H2O2-consuming peroxidase (guaiacol POD) in the apoplastic washing fluid (AWF) of leaves for early stages of Mn toxicity and genotypic differences in Mn tolerance of cowpea. Leaf AWF of the Mn-sensitive cultivar (cv) TVu 91 but not of the Mn-tolerant cv 1987 showed an increase of guaiacol-POD and NADH-peroxidase activities at elevated AWF Mn concentrations. two-dimensional resolutions of AWF proteins revealed that cv TVu 91 expressed more and additional proteins at high Mn treatment, whereas Mn-tolerant cv TVu 1987 remained nearly unaffected. In both cultivars, NADH-peroxidase activity and accompanied H2O2 formation rate in vitro were significantly affected by Mn2+, p-coumaric acid, and metabolites occurring in the AWF. The total phenol concentration in the AWF was indicative of advanced stages of Mn toxicity but was rather unrelated to early stages of Mn toxicity and genotypic differences in Mn tolerance. The NADH oxidation by AWF PODs was significantly delayed or enhanced in the presence of the protein-free AWF from cv TVu 1987 or cv TVu 91, respectively. High-performance liquid chromatography analysis of AWF indicates the presence of phenols in cv TVu 1987 not observed in cv TVu 91. We conclude from our studies that the H2O2-producing NADH peroxidase and its modulation by stimulating or inhibiting phenolic compounds in the leaf apoplast play a major role for Mn toxicity and Mn tolerance in cowpea.