Phosphorylation by CIPK23 regulates the high-affinity Mn transporter NRAMP1 in Arabidopsis.

Manganese (Mn) is essential for plants but is toxic when taken up in excess. To maintain Mn homeostasis, the root Mn transporter natural resistance associated macrophage protein 1 (NRAMP1) cycles from the plasma membrane to endosomes upon phosphorylation. To identify the kinase involved, a split-luciferase screening was carried out between NRAMP1 and kinases of the CIPK family and identified CIPK23 as a partner of NRAMP1. The interaction was confirmed by split-mCitrine bimolecular fluorescence complementation and co-immunoprecipitation assays. In vitro phosphorylation assays pinpointed two CIPK23 target residues in NRAMP1, among which serine 20, important for endocytosis. Interestingly, Mn-induced internalization of NRAMP1 was unaffected by cipk23 mutation suggesting a potential redundancy between CIPK23 and other kinase(s). How CIPK23 could regulate NRAMP1 in response to Mn availability is discussed.

Study of the Stability, Uptake and Transformations of Zero Valent Iron Nanoparticles in a Model Plant by Means of an Optimised Single Particle ICP-MS/MS Method.

In the context of the widespread distribution of zero valent iron nanoparticles (nZVI) in the environment and its possible exposure to many aquatic and terrestrial organisms, this study investigates the effects, uptake, bioaccumulation, localisation and possible transformations of nZVI in two different forms (aqueous dispersion-Nanofer 25S and air-stable powder-Nanofer STAR) in a model plant-Arabidopsis thaliana. Seedlings exposed to Nanofer STAR displayed symptoms of toxicity, including chlorosis and reduced growth. At the tissue and cellular level, the exposure to Nanofer STAR induced a strong accumulation of Fe in the root intercellular spaces and in Fe-rich granules in pollen grains. Nanofer STAR did not undergo any transformations during 7 days of incubation, while in Nanofer 25S, three different behaviours were observed: (i) stability, (ii) partial dissolution and (iii) the agglomeration process. The size distributions obtained by SP-ICP-MS/MS demonstrated that regardless of the type of nZVI used, iron was taken up and accumulated in the plant, mainly in the form of intact nanoparticles. The agglomerates created in the growth medium in the case of Nanofer 25S were not taken up by the plant. Taken together, the results indicate that Arabidopsis plants do take up, transport and accumulate nZVI in all parts of the plants, including the seeds, which will provide a better understanding of the behaviour and transformations of nZVI once released into the environment, a critical issue from the point of view of food safety.

NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis.

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.

Golgi in and out: multifaceted role and journey of manganese.

Manganese (Mn) is pivotal for plant growth and development but little is known about the processes that control its homeostasis in the cell. A spotlight on the pools of intracellular manganese and their cellular function has recently been gained through the characterization of new Mn transporters. In particular, transporters catalyzing the ins and outs of Mn at the various Golgi membranes have revealed the central role of the Golgi pool of Mn in the synthesis of the cell wall and as a reservoir for the numerous cellular Mn-dependent pathways whose calibration relies on a set of Golgi-resident transporters of the BICAT and NRAMP families.

MCO1 and MCO3, two putative ascorbate oxidases with ferroxidase activity, new candidates for the regulation of apoplastic iron excess in Arabidopsis.

Iron (Fe) is an essential metal ion that plays a major role as a cofactor in many biological processes. The balance between the Fe2+ and Fe3+ forms is central for cellular Fe homeostasis because it regulates its transport, utilization, and storage. Contrary to Fe3+ reduction that is crucial for Fe uptake by roots in deficiency conditions, ferroxidation has been much less studied. In this work, we have focused on the molecular characterization of two members of the MultiCopper Oxidase family (MCO1 and MCO3) that share high identity with the Saccharomyces cerevisiae ferroxidase Fet3. The heterologous expression of MCO1 and MCO3 restored the growth of the yeast fet3fet4 mutant, impaired in high and low affinity Fe uptake and otherwise unable to grow in Fe deficient media, suggesting that MCO1 and MCO3 were functional ferroxidases. The ferroxidase enzymatic activity of MCO3 was further confirmed by the measurement of Fe2+-dependent oxygen consumption, because ferroxidases use oxygen as electron acceptor to generate water molecules. In planta, the expression of MCO1 and MCO3 was induced by increasing Fe concentrations in the medium. Promoter-GUS reporter lines showed that MCO1 and MCO3 were mostly expressed in shoots and histochemical analyses further showed that both promoters were highly active in mesophyll cells. Transient expression of MCO1-RFP and MCO3-RFP in tobacco leaves revealed that both proteins were localized in the apoplast. Moreover, cell plasmolysis experiments showed that MCO1 remained closely associated to the plasma membrane whereas MCO3 filled the entire apoplast compartment. Although the four knock out mutant lines isolated (mco1-1, mco1-2, mco3-1, and mco3-2) did not display any macroscopic phenotype, histochemical staining of Fe with the Perls/DAB procedure revealed that mesophyll cells of all four mutants overaccumulated Fe inside the cells in Fe-rich structures in the chloroplasts, compared with wild-type. These results suggested that the regulation of Fe transport in mesophyll cells had been disturbed in the mutants, in both standard condition and Fe excess. Taken together, our findings strongly suggest that MCO1 and MCO3 participate in the control of Fe transport in the mesophyll cells, most likely by displacing the Fe2+/Fe3+ balance toward Fe3+ in the apoplast and therefore limiting the accumulation of Fe2+, which is more mobile and prone to be transported across the plasma membrane.

Towards routine 3D characterization of intact mesoscale samples by multi-scale and multimodal scanning X-ray tomography.

Non-invasive multi-scale and multimodal 3D characterization of heterogeneous or hierarchically structured intact mesoscale samples is of paramount importance in tackling challenging scientific problems. Scanning hard X-ray tomography techniques providing simultaneous complementary 3D information are ideally suited to such studies. However, the implementation of a robust on-site workflow remains the bottleneck for the widespread application of these powerful multimodal tomography methods. In this paper, we describe the development and implementation of such a robust, holistic workflow, including semi-automatic data reconstruction. Due to its flexibility, our approach is especially well suited for on-the-fly tuning of the experiments to study features of interest progressively at different length scales. To demonstrate the performance of the method, we studied, across multiple length scales, the elemental abundances and morphology of two complex biological systems, Arabidopsis plant seeds and mouse renal papilla samples. The proposed approach opens the way towards routine multimodal 3D characterization of intact samples by providing relevant information from pertinent sample regions in a wide range of scientific fields such as biology, geology, and material sciences.

B3 Transcription Factors Determine Iron Distribution and FERRITIN Gene Expression in Embryo but Do Not Control Total Seed Iron Content.

Iron is an essential micronutrient for humans and other organisms. Its deficiency is one of the leading causes of anemia worldwide. The world health organization has proposed that an alternative to increasing iron content in food is through crop biofortification. One of the most consumed part of crops is the seed, however, little is known about how iron accumulation in seed occurs and how it is regulated. B3 transcription factors play a critical role in the accumulation of storage compounds such as proteins and lipids. Their role in seed maturation has been well characterized. However, their relevance in accumulation and distribution of micronutrients like iron remains unknown. In Arabidopsis thaliana and other plant models, three master regulators belonging to the B3 transcription factors family have been identified: FUSCA3 (FUS3), LEAFY COTYLEDON2 (LEC2), and ABSCISIC ACID INSENSITIVE 3 (ABI3). In this work, we studied how seed iron homeostasis is affected in B3 transcription factors mutants using histological and molecular approaches. We determined that iron distribution is modified in abi3, lec2, and fus3 embryo mutants. For abi3-6 and fus3-3 mutant embryos, iron was less accumulated in vacuoles of cells surrounding provasculature compared with wild type embryos. lec2-1 embryos showed no difference in the pattern of iron distribution in hypocotyl, but a dramatic decrease of iron was observed in cotyledons. Interestingly, for the three mutant genotypes, total iron content in dry mutant seeds showed no difference compared to wild type. At the molecular level, we showed that genes encoding the iron storage ferritins proteins are misregulated in mutant seeds. Altogether our results support a role of the B3 transcription factors ABI3, LEC2, and FUS3 in maintaining iron homeostasis in Arabidopsis embryos.

NRAMP6 and NRAMP1 cooperatively regulate root growth and manganese translocation under manganese deficiency in Arabidopsis.

The essential micronutrient manganese (Mn) in plants regulates multiple biological processes including photosynthesis and oxidative stress. Some Natural Resistance-Associated Macrophage Proteins (NRAMPs) have been reported to play critical roles in Mn uptake and reutilization in low Mn conditions. NRAMP6 was demonstrated to regulate cadmium tolerance and iron utilization in Arabidopsis. Nevertheless, it is unclear whether NRAMP6 plays a role in Mn nutrition. Here, we report that NRAMP6 cooperates with NRAMP1 in Mn utilization. Mutation of NRAMP6 in nramp1 but not in a wild-type background reduces root growth and Mn translocation from the roots to shoots under Mn deficient conditions. Grafting experiments revealed that NRAMP6 expression in both the roots and shoots is required for root growth and Mn translocation under Mn deficiency. We also showed that NRAMP1 could replace NRAMP6 to sustain root growth under Mn deficiency, but not vice versa. Mn deficiency does not affect the transcript level of NRAMP6, but is able to increase and decrease the protein accumulation of NRAMP6 in roots and shoots, respectively. Furthermore, NRAMP6 can be localized to both the plasma membrane and endomembranes including the endoplasmic reticulum, and Mn deficiency enhances the localization of NRAMP6 to the plasma membrane in Arabidopsis plants. NRAMP6 could rescue the defective growth of the yeast mutant Δsmf2, which is deficient in endomembrane Mn transport. Our results reveal the important role of NRAMP6 in Mn nutrition and in the long-distance signaling between the roots and shoots under Mn deficient conditions.

AtDTX25, a member of the multidrug and toxic compound extrusion family, is a vacuolar ascorbate transporter that controls intracellular iron cycling in Arabidopsis.

Iron (Fe) is an essential element, its transport is regulated by the cell redox balance. In seeds, Fe enters the embryo as Fe2+ and is stored in vacuoles as Fe3+ . Through its ferric reduction activity, ascorbate plays a major role in Fe redox state and therefore Fe transport within the seed. We searched for ascorbate membrane transporters responsible for controlling Fe reduction by screening the yeast ferric reductase-deficient fre1 strain and isolated AtDTX25, a member of the Multidrug And Toxic compound Extrusion (MATE) family. AtDTX25 was shown to mediate ascorbate efflux when expressed in yeast and Xenopus oocytes, in a pH-dependent manner. In planta, AtDTX25 is highly expressed during germination and encodes a vacuolar membrane protein. Isolated vacuoles from AtDTX25-1 knockout mutant contained less ascorbate and more Fe than wild-type (WT), and mutant seedlings were highly sensitive to Fe deficiency. Iron imaging further showed that the remobilisation of Fe from vacuoles was highly impaired in mutant seedlings. Taken together, our results established AtDTX25 as a vacuolar ascorbate transporter, required during germination to promote the reduction of the pool of stored Fe3+ and its remobilisation to feed the developing seedling.

Manganese triggers phosphorylation-mediated endocytosis of the Arabidopsis metal transporter NRAMP1.

The NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1) transporter guarantees plant survival of manganese (Mn) deficiency by mediating Mn entry into root cells. Unlike other high-affinity metal transporters, NRAMP1 is only slightly regulated at the transcriptional level. We show here that adequate Mn content in tissues is safeguarded through a tight control of the quantity of NRAMP1 present at the surface of root cells. Depending on Mn availability, an NRAMP1-GFP fusion protein cycles dynamically between the plasma membrane (PM) and endosomal compartments. This involves a clathrin-mediated endocytosis pathway, as disrupting this pathway in auxilin-overexpressor lines prevents NRAMP1 internalization. Mutation of the phosphorylated serine residues 20, 22 and 24 in the cytosol-exposed N terminus of NRAMP1 alters its membrane distribution. Indeed, a phospho-dead mutation stabilizes NRAMP1 at the PM, regardless of the Mn regime, and dramatically reduces plant tolerance to Mn toxicity. Conversely a phosphomimetic mutant is constitutively internalized into endosomes. Together, these data establish that phosphorylation of NRAMP1 is the trigger for its Mn-induced endocytosis and represents the main level of regulation of this transporter. Furthermore, the extent of Mn toxicity observed when interrupting NRAMP1 membrane cycling undermines the dogma that Mn is only marginally toxic to plants.

Paspalum urvillei and Setaria parviflora, two grasses naturally adapted to extreme iron-rich environments.

Paspalum urvillei and Setaria parviflora are two plant species naturally adapted to iron-rich environments such as around iron mines wastes. The aim of our work was to characterize how these two species cope with these extreme conditions by comparing them with related model species, Oryza sativa and Setaria viridis, that appeared to be much less tolerant to Fe excess. Both Paspalum urvillei and Setaria parviflora were able to limit the amount of Fe accumulated within roots and shoots, compared to the less tolerant species. Perls/DAB staining of Fe in root cross sections indicated that Paspalum urvillei and Setaria parviflora responded through the build-up of the iron plaque (IP), suggesting a role of this structure in the limitation of Fe uptake. Synchrotron µXRF analyses showed the presence of phosphorus, calcium, silicon and sulfur on IP of Paspalum urvillei roots and µXANES analyses identified Fe oxyhydroxide (ferrihydrite) as the main Fe form. Once within roots, high concentrations of Fe were localized in the cell walls and vacuoles of Paspalum urvillei, Setaria parviflora and O. sativa whereas Setaria viridis accumulated Fe in ferritins. The Fe forms translocated to the shoots of Setaria parviflora were identified as tri-iron complexes with citrate and malate. In leaves, all species accumulated Fe in the vacuoles of bundle sheath cells and as ferritin complexes in plastids. Taken together, our results strongly suggest that Paspalum urvillei and Setaria parviflora set up mechanisms of Fe exclusion in roots and shoots to limit the toxicity induced by Fe excess.

Split green fluorescent protein as a tool to study infection with a plant pathogen, Cauliflower mosaic virus.

The split GFP technique is based on the auto-assembly of GFP when two polypeptides-GFP1-10 (residues 1-214; the detector) and GFP11 (residues 215-230; the tag)-both non-fluorescing on their own, associate spontaneously to form a fluorescent molecule. We evaluated this technique for its efficacy in contributing to the characterization of Cauliflower mosaic virus (CaMV) infection. A recombinant CaMV with GFP11 fused to the viral protein P6 (a key player in CaMV infection and major constituent of viral factory inclusions that arise during infection) was constructed and used to inoculate transgenic Arabidopsis thaliana expressing GFP1-10. The mutant virus (CaMV11P6) was infectious, aphid-transmissible and the insertion was stable over many passages. Symptoms on infected plants were delayed and milder. Viral protein accumulation, especially of recombinant 11P6, was greatly decreased, impeding its detection early in infection. Nonetheless, spread of infection from the inoculated leaf to other leaves was followed by whole plant imaging. Infected cells displayed in real time confocal laser scanning microscopy fluorescence in wild type-looking virus factories. Thus, it allowed for the first time to track a CaMV protein in vivo in the context of an authentic infection. 11P6 was immunoprecipitated with anti-GFP nanobodies, presenting a new application for the split GFP system in protein-protein interaction assays and proteomics. Taken together, split GFP can be an attractive alternative to using the entire GFP for protein tagging.

Copper transporter COPT5 participates in the crosstalk between vacuolar copper and iron pools mobilisation.

Copper (Cu) deficiency affects iron (Fe) homeostasis in several plant processes, including the increased Fe requirements due to cuproprotein substitutions for the corresponding Fe counterpart. Loss-of-function mutants from Arabidopsis thaliana high affinity copper transporter COPT5 and Fe transporters NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 3/4 (NRAMP3 and NRAMP4) were used to study the interaction between metals internal pools. A physiological characterisation showed that the copt5 mutant is sensitive to Fe deficiency, and that nramp3nramp4 mutant growth was severely affected under limiting Cu. By a transcriptomic analysis, we observed that NRAMP4 expression was highly induced in the copt5 mutant under Cu deficiency, while COPT5 was overexpressed in the nramp3nramp4 mutant. As a result, an enhanced mobilisation of the vacuolar Cu or Fe pools, when the other metal export through the tonoplast is impaired in the mutants, has been postulated. However, metals coming from internal pools are not used to accomplish the increased requirements that derive from metalloprotein substitution under metal deficiencies. Instead, the metal concentrations present in aerial parts of the copt5 and nramp3nramp4 mutants conversely show compensated levels of these two metals. Together, our data uncover an interconnection between Cu and Fe vacuolar pools, whose aim is to fulfil interorgan metal translocation.

Intracellular Distribution of Manganese by the Trans-Golgi Network Transporter NRAMP2 Is Critical for Photosynthesis and Cellular Redox Homeostasis.

Plants require trace levels of manganese (Mn) for survival, as it is an essential cofactor in oxygen metabolism, especially O2 production via photosynthesis and the disposal of superoxide radicals. These processes occur in specialized organelles, requiring membrane-bound intracellular transporters to partition Mn between cell compartments. We identified an Arabidopsis thaliana member of the NRAMP family of divalent metal transporters, NRAMP2, which functions in the intracellular distribution of Mn. Two knockdown alleles of NRAMP2 showed decreased activity of photosystem II and increased oxidative stress under Mn-deficient conditions, yet total Mn content remained unchanged. At the subcellular level, these phenotypes were associated with a loss of Mn content in vacuoles and chloroplasts. NRAMP2 was able to rescue the mitochondrial yeast mutant mtm1∆ In plants, NRAMP2 is a resident protein of the trans-Golgi network. NRAMP2 may act indirectly on downstream organelles by building up a cytosolic pool that is used to feed target compartments. Moreover, not only does the nramp2 mutant accumulate superoxide ions, but NRAMP2 can functionally replace cytosolic superoxide dismutase in yeast, indicating that the pool of Mn displaced by NRAMP2 is required for the detoxification of reactive oxygen species.

Phosphatidylinositol 3-phosphate-binding protein AtPH1 controls the localization of the metal transporter NRAMP1 in Arabidopsis.

"Too much of a good thing" perfectly describes the dilemma that living organisms face with metals. The tight control of metal homeostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings require the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient phenotype of nramp3nramp4 Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 function leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain protein as a regulator of plant metal transporter localization, providing evidence that PH domain proteins may be effectors of PI3P for protein sorting.

New routes for plant iron mining.

Contents 521 I. 521 II. 522 III. 523 IV. 524 525 References 525 SUMMARY: Plant iron (Fe) uptake relies to a large extent on the capacity of cells to control and extract Fe pools safely conserved in extracytoplasmic environments such as the apoplast and vacuoles, at least as much as on the transport machinery nested in plasma membranes. Recent studies on root and embryo Fe nutrition support this assertion and show that the root Fe-deficiency response also includes the dynamic use of a large Fe reservoir bound to cell wall components in the root apoplast, secretion in the apoplast of phenolic compounds of the coumarin family, which solubilize Fe in calcareous soils, and inhibition of suberization of endodermal cells in order to allow apoplastic and transcellular radial transport of Fe. All of these responses are regulated by the stress hormones ethylene and abscisic acid (ABA), suggesting an integrated strategy within the root to adapt to Fe shortage. For its nutrition, the embryo has developed both an original uptake mechanism, in which ascorbate is effluxed to chemically reduce Fe3+ to the transport-competent Fe2+ form, and an efficient strategy to control utilization of a large Fe pool in vacuoles. This review will attempt to summarize exciting new insights into the diverse routes that Fe takes to feed plant tissues.

Systems-wide analysis of manganese deficiency-induced changes in gene activity of Arabidopsis roots.

Manganese (Mn) is pivotal for plant growth and development, but little information is available regarding the strategies that evolved to improve Mn acquisition and cellular homeostasis of Mn. Using an integrated RNA-based transcriptomic and high-throughput shotgun proteomics approach, we generated a comprehensive inventory of transcripts and proteins that showed altered abundance in response to Mn deficiency in roots of the model plant Arabidopsis. A suite of 22,385 transcripts was consistently detected in three RNA-seq runs; LC-MS/MS-based iTRAQ proteomics allowed the unambiguous determination of 11,606 proteins. While high concordance between mRNA and protein expression (R = 0.87) was observed for transcript/protein pairs in which both gene products accumulated differentially upon Mn deficiency, only approximately 10% of the total alterations in the abundance of proteins could be attributed to transcription, indicating a large impact of protein-level regulation. Differentially expressed genes spanned a wide range of biological functions, including the maturation, translation, and transport of mRNAs, as well as primary and secondary metabolic processes. Metabolic analysis by UPLC-qTOF-MS revealed that the steady-state levels of several major glucosinolates were significantly altered upon Mn deficiency in both roots and leaves, possibly as a compensation for increased pathogen susceptibility under conditions of Mn deficiency.

The high-affinity metal Transporters NRAMP1 and IRT1 Team up to Take up Iron under Sufficient Metal Provision.

Iron (Fe) and manganese (Mn) are essential metals which, when scarce in the growth medium, are respectively taken up by the root high affinity transporters IRT1 and NRAMP1 in Arabidopsis thaliana. The molecular bases for low affinity transport however remained unknown. Since IRT1 and NRAMP1 have a broad range of substrates among metals, we tested the hypothesis that they might be functionally redundant by generating nramp1 irt1 double mutants. These plants showed extreme Fe-deficiency symptoms despite optimal provision of the metal. Their phenotype, which includes low Fe and Mn contents and a defect of Fe entry into root cells as revealed by Fe staining, is rescued by high Fe supply. Using a promoter swap-based strategy, we showed that root endodermis retains the ability to carry out high affinity Fe transport and furthermore might be important to high-affinity Mn uptake. We concluded that NRAMP1 plays a pivotal role in Fe transport by cooperating with IRT1 to take up Fe in roots under replete conditions, thus providing the first evidence for a low affinity Fe uptake system in plants.