In Vivo Assimilation of CuS, Iron Oxide and Iron Oxide@CuS Nanoparticles in Mice: A 6-Month Follow-Up Study

Nanoparticles (NPs) are at the leading edge of nanomedicine, and determining their biosafety remains a mandatory precondition for biomedical applications. Herein, we explore the bioassimilation of copper sulfide NPs reported as powerful photo-responsive anticancer therapeutic agents. The nanoparticles investigated present a hollow shell morphology, that can be left empty (CuS NPs) or be filled with an iron oxide flower-like core (iron oxide@CuS NPs), and are compared with the iron oxide nanoparticles only (iron oxide NPs). CuS, iron oxide@CuS and iron oxide NPs were injected in 6-week-old mice, at doses coherent with an antitumoral treatment. Cu and Fe were quantified in the liver, spleen, kidneys, and lungs over 6 months, including the control animals, thus providing endogenous Cu and Fe levels in the first months after animal birth. After intravenous NPs administration, 77.0 ± 3.9% of the mass of Cu injected, and 78.6 ± 3.8% of the mass of Fe, were detected in the liver. In the spleen, we found 3.3 ± 0.6% of the injected Cu and 3.8 ± 0.6% for the Fe. No negative impact was observed on organ weight, nor on Cu or Fe homeostasis in the long term. The mass of the two metals returned to the control values within three months, a result that was confirmed by transmission electron microscopy and histology images. This bioassimilation with no negative impact comforts the possible translation of these nanomaterials into clinical practice.

The Effect of the Upregulated Expression of Fe Regulators IMA1 and bHLH104 in Arabidopsis on the Root Length and Fe Homeostasis Under Pi Starvation

Phosphate (Pi) and iron (Fe) are two essential mineral nutrients for plant growth and development. Pi starvation triggers the Fe local redistribution and over-accumulation, resulting in the reduction of the primary root, while represses the expression of Fe uptake genes. Nevertheless, the antagonistic mechanism between P and Fe nutrition in plant remain not addressed. Here, the effect of the upregulated expression of Fe regulators IMA1 and bHLH104 driven by the different-type promoters (proCaMV 35S, the promoters of Pi-starvation responsive genes proIPS1 and proPHT1;4) in response to Pi starvation was investigated in Arabidopsis. The results showed that the expression of Fe uptake genes IRT1 and FRO2 was successfully upregulated in proIPS1::IMA1, proPHT1;4::IMA1 and proIPS1::bHLH104 under Pi starvation while decreased in pro35S::IMA1, pro35S::bHLH104 and proPHT1;4::bHLH104, compared with that in the corresponding plants under Pi sufficiency. Although the length and Fe distribution in roots of them didn’t have significant difference with wild type under Pi starvation, the Fe distribution and total Fe contents were significantly increased in shoots of proIPS1::IMA1, proPHT1;4::IMA1 and proIPS1::bHLH104 while were decreased in proPHT1;4::bHLH104. The higher Fe concentrations in the Pi-starved transgenic plants also conferred the obviously tolerance to Fe deficiency. Their biomasses and total P concentrations showed no difference with wild type, regardless of Pi sufficiency or deficiency. Therefore, this approach would be a novel manipulation to modify Fe nutrient via coupling with Pi starvation in plants.

Analysis of the VIT1 Promoter Activity in Developing Arabidopsis thaliana Plants

Iron (Fe) deficiency in plants is one of the widespread problems limiting agricultural production. Generating crops more tolerant to Fe deficiency by genetic engineering or breeding is of great interest but challenging due to the knowledge gaps in general plant Fe homeostasis. Although several genes involved in Fe homeostasis have been identified, characterization of their roles is mainly limited to specific organs at specific developmental stages of the plant, where their mutants show the most striking phenotype. Vacuolar Iron Transporter 1 (VIT1) is a well-known gene that has been characterized for its function in the mature seed of Arabidopsis thaliana. VIT1 is an Fe transporter that determines the correct distribution of Fe storage in this organ. The study aimed to explore new physiological functions for VIT1. As a first step, Arabidopsis thaliana plants that contain PromoterVIT1: GUS constructs were used to study the temporal and spatial expression of the gene throughout the plant’s lifecycle. GUS histochemical staining revealed the VIT1 promoter is active in the mature leaves and mature reproductive organs. VIT1 promoter activity in the stamen increased developmentally and was limited to tapetum and guard cells in the pollen sac. In the female organ, VIT1 promoter activity increased as the pistil developed into a silique. Although all the silique exhibited staining, staining density was higher in the peduncle, replum, and stigma regions. Inside the developing silique, funicles were heavily stained. Furthermore, in silico analyses of VT1 transcriptome and protein levels confirmed flower and the silique are hot spots for VT1 activity. Thus, the results may suggest a possible involvement of VT1 protein in several stages of the reproductive system, specifically in the flowering and in the fruit development.

Cross-Talks Between Macro- and Micronutrient Uptake and Signaling in Plants

In nature, land plants as sessile organisms are faced with multiple nutrient stresses that often occur simultaneously in soil. Nitrogen (N), phosphorus (P), sulfur (S), zinc (Zn), and iron (Fe) are five of the essential nutrients that affect plant growth and health. Although these minerals are relatively inaccessible to plants due to their low solubility and relative immobilization, plants have adopted coping mechanisms for survival under multiple nutrient stress conditions. The double interactions between N, Pi, S, Zn, and Fe have long been recognized in plants at the physiological level. However, the molecular mechanisms and signaling pathways underlying these cross-talks in plants remain poorly understood. This review preliminarily examined recent progress and current knowledge of the biochemical and physiological interactions between macro- and micro-mineral nutrients in plants and aimed to focus on the cross-talks between N, Pi, S, Zn, and Fe uptake and homeostasis in plants. More importantly, we further reviewed current studies on the molecular mechanisms underlying the cross-talks between N, Pi, S, Zn, and Fe homeostasis to better understand how these nutrient interactions affect the mineral uptake and signaling in plants. This review serves as a basis for further studies on multiple nutrient stress signaling in plants. Overall, the development of an integrative study of multiple nutrient signaling cross-talks in plants will be of important biological significance and crucial to sustainable agriculture.

Transcriptomic analysis revealed reactive oxygen species scavenging mechanisms associated with ferrous iron toxicity in aromatic Keteki Joha rice

Lowland acidic soils with water-logged regions are often affected by ferrous iron (Fe2+) toxicity, a major yield-limiting factor of rice production. The Reactive Oxygen Species (ROS) was hypothesized to be crucial under severe Fe2+ toxicity conditions. However, molecular mechanisms and associated ROS homeostasis genes are still not well-explored. In this study, a comparative RNA-Seq based transcriptome analysis was conducted to understand the Fe2+ toxicity tolerance mechanism in aromatic Keteki Joha. About 69 Fe homeostasis related genes and their homologs were identified, where most of the genes were downregulated. Differentially expressed genes (DEGs) are associated with biological processes- response to stress, stimulus and abiotic stimulus. DEGs involved in the Biosynthesis of amino acids, RNA degradation, Glutathione metabolism etc. were induced, whereas, Phenylpropanoid biosynthesis, Photosynthesis, and Fatty acid elongation were inhibited. The Mitochondrial iron transporter (OsMIT), Vacuolar Iron Transporter 2 (OsVIT2), Ferritin (OsFER), Vacuolar Mugineic Acid Transporter (OsVMT), Phenolic Efflux Zero1 (OsPEZ1), Root Meander Curling (OsRMC), Nicotianamine synthase (OsNAS3), etc. were upregulated in different tissues suggesting the importance of Fe retention and sequestration for detoxification. However, several antioxidants, ROS scavenging genes and abiotic stress-responsive transcription factors indicate ROS homeostasis as one of the most important defense mechanisms under severe Fe2+ toxicity. The CAT, GSH, APX, MDHAR, DHAR, GR were upregulated. Moreover, abiotic stress-responsive transcription factors NAC, MYB, ARF, bZIP, WRKY, C2H2-ZFP were also upregulated. Accordingly, ROS homeostasis has been proposed to be an important defense mechanism under such conditions. Thus, our results will enrich the knowledge of understanding Fe-homeostasis in rice.

Involvement of Arabidopsis Multi-Copper Oxidase-Encoding LACCASE12 in Root-to-Shoot Iron Partitioning: A Novel Example of Copper-Iron Crosstalk

Numerous central biological processes depend on the participation of the essential elements iron (Fe) or copper (Cu), including photosynthesis, respiration, cell wall remodeling and oxidative stress protection. Yet, both Fe and Cu metal cations can become toxic when accumulated in excess. Because of the potent ligand-binding and redox chemistries of these metals, there is a need for the tight and combined homeostatic control of their uptake and distribution. Several known examples pinpoint an inter-dependence of Fe and Cu homeostasis in eukaryotes, mostly in green algae, yeast and mammals, but this is less well understood in multicellular plants to date. In Arabidopsis, Cu deficiency causes secondary Fe deficiency, and this is associated with reduced in vitro ferroxidase activity and decreased root-to-shoot Fe translocation. Here we summarize the current knowledge of the cross-talk between Cu and Fe homeostasis and present a partial characterization of LACCASE12 (LAC12) that encodes a member of the multicopper oxidase (MCO) protein family in Arabidopsis. LAC12 transcript levels increase under Fe deficiency. The phenotypic characterization of two mutants carrying T-DNA insertions suggests a role of LAC12 in root-to-shoot Fe partitioning and in maintaining growth on Fe-deficient substrates. A molecular understanding of the complex interactions between Fe and Cu will be important for combating Fe deficiency in crops and for advancing biofortification approaches.

Nitric Oxide Prevents Fe Deficiency-Induced Photosynthetic Disturbance, and Oxidative Stress in Alfalfa by Regulating Fe Acquisition and Antioxidant Defense

Iron (Fe) deficiency impairs photosynthetic efficiency, plant growth and biomass yield. This study aimed to reveal the role of nitric oxide (NO) in restoring Fe-homeostasis and oxidative status in Fe-deficient alfalfa. In alfalfa, a shortage of Fe negatively affected the efficiency of root andshoot length, leaf greenness, maximum quantum yield PSII (Fv/Fm), Fe, S, and Zn accumulation, as well as an increase in H2O2 accumulation. In contrast, in the presence of sodium nitroprusside (SNP), a NO donor, these negative effects of Fe deficiency were largely reversed. In response to the SNP, the expression of Fe transporters (IRT1, NRAMP1) and S transporter (SULTR1;2) genes increased in alfalfa. Additionally, the detection of NO generation using fluorescence microscope revealed that SNP treatment increased the level of NO signal, indicating that NO may act as regulatory signal in response to SNP in plants. Interestingly, the increase of antioxidant genes and their related enzymes (Fe-SOD, APX) in response to SNP treatment suggests that Fe-SOD and APX are key contributors to reducing ROS (H2O2) accumulation and oxidative stress in alfalfa. Furthermore, the elevation of Ascorbate-glutathione (AsA-GSH) pathway-related genes (GR and MDAR) Fe-deficiency with SNP implies that the presence of NO relates to enhanced antioxidant defense against Fe-deficiency stress.

Salmonella Typhimurium and Pseudomonas aeruginosa Respond Differently to the Fe Chelator Deferiprone and to Some Novel Deferiprone Derivatives

The ability to obtain Fe is critical for pathogens to multiply in their host. For this reason, there is significant interest in the identification of compounds that might interfere with Fe management in bacteria. Here we have tested the response of two Gram-negative pathogens, Salmonella enterica serovar Typhimurium (STM) and Pseudomonas aeruginosa (PAO1), to deferiprone (DFP), a chelating agent already in use for the treatment of thalassemia, and to some DFP derivatives designed to increase its lipophilicity. Our results indicate that DFP effectively inhibits the growth of PAO1, but not STM. Similarly, Fe-dependent genes of the two microorganisms respond differently to this agent. DFP is, however, capable of inhibiting an STM strain unable to synthesize enterochelin, while its effect on PAO1 is not related to the capability to produce siderophores. Using a fluorescent derivative of DFP we have shown that this chelator can penetrate very quickly into PAO1, but not into STM, suggesting that a selective receptor exists in Pseudomonas. Some of the tested derivatives have shown a greater ability to interfere with Fe homeostasis in STM compared to DFP, whereas most, although not all, were less active than DFP against PAO1, possibly due to interference of the added chemical tails with the receptor-mediated recognition process. The results reported in this work indicate that DFP can have different effects on distinct microorganisms, but that it is possible to obtain derivatives with a broader antimicrobial action.

IRON MAN interacts with BRUTUS to maintain iron homeostasis in Arabidopsis

IRON MAN (IMA) peptides, a family of small peptides, control iron (Fe) transport in plants, but their roles in Fe signaling remain unclear. BRUTUS (BTS) is a potential Fe sensor that negatively regulates Fe homeostasis by promoting the ubiquitin-mediated degradation of bHLH105 and bHLH115, two positive regulators of the Fe deficiency response. Here, we show that IMA peptides interact with BTS. The C-terminal parts of IMA peptides contain a conserved BTS interaction domain (BID) that is responsible for their interaction with the C terminus of BTS. Arabidopsis thaliana plants constitutively expressing IMA genes phenocopy the bts-2 mutant. Moreover, IMA peptides are ubiquitinated and degraded by BTS. bHLH105 and bHLH115 also share a BID, which accounts for their interaction with BTS. IMA peptides compete with bHLH105/bHLH115 for interaction with BTS, thereby inhibiting the degradation of these transcription factors by BTS. Genetic analyses suggest that bHLH105/bHLH115 and IMA3 have additive roles and function downstream of BTS. Moreover, the transcription of both BTS and IMA3 is activated directly by bHLH105 and bHLH115 under Fe-deficient conditions. Our findings provide a conceptual framework for understanding the regulation of Fe homeostasis: IMA peptides protect bHLH105/bHLH115 from degradation by sequestering BTS, thereby activating the Fe deficiency response.