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.

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.

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.

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.

Over-expression of the bacterial phytase US417 in Arabidopsis reduces the concentration of phytic acid and reveals its involvement in the regulation of sulfate and phosphate homeostasis and signaling.

Phytic acid (PA) is the main phosphorus storage form in plant seeds. It is recognized as an anti-nutrient for humans and non-ruminant animals, as well as one of the major sources of phosphorus that contributes to eutrophication. Therefore, engineering plants with low PA content without affecting plant growth capacity has become a major focus in plant breeding. Nevertheless, lack of knowledge on the role of PA seed reserves in regulating plant growth and in maintaining ion homeostasis hinders such an agronomical application. In this context, we report here that the over-expression of the bacterial phytase PHY-US417 in Arabidopsis leads to a significant decrease in seed PA, without any effect on the seed germination potential. Interestingly, this over-expression also induced a higher remobilization of free iron during germination. Moreover, the PHY-over-expressor lines show an increase in inorganic phosphate and sulfate contents, and a higher biomass production after phosphate starvation. Finally, phosphate sensing was altered because of the changes in the expression of genes induced by phosphate starvation or involved in phosphate or sulfate transport. Together, these results show that the over-expression of PHY-US417 reduces PA concentration, and provide the first evidence for the involvement of PA in the regulation of sulfate and phosphate homeostasis and signaling.