Search

Cell-to-cell communication plays a cardinal role in the biology of multicellular organisms. H2O2 is an important cell-to-cell signaling molecule involved in the response of mammalian cells to wounding and other stimuli. We previously identified a signaling pathway that transmits wound-induced cell-to-cell H2O2 signals within minutes over long distances, measured in centimeters, in a monolayer of cardiomyocytes. Here we report that this long-distance H2O2 signaling pathway is accompanied by enhanced accumulation of cytosolic H2O2 and altered redox state in cells along its path. We further show that it requires the production of superoxide, as well as the function of gap junctions, and that it is accompanied by changes in the abundance of hundreds of proteins in cells along its path. Our findings highlight the existence of a unique and rapid long-distance H2O2 signaling pathway that could play an important role in different inflammatory responses, wound responses/healing, cardiovascular disease, and/or other conditions.

Abstract Cell Penetrating Peptides (CPPs) are promising anticancer and antimicrobial drugs. We recently reported that a peptide derived from the human mitochondrial/ER membrane-anchored NEET protein, Nutrient Autophagy Factor 1 (NAF-1; NAF-144-67), selectively permeates and kills human metastatic epithelial breast cancer cells (MDA-MB-231), but not control epithelial cells. As cancer cells alter their phenotype during growth and metastasis, we tested whether NAF-144–67 would also be efficient in killing other human epithelial breast cancer cells that may have a different phenotype. Here we report that NAF-144–67 is efficient in killing BT-549, Hs 578T, MDA-MB-436, and MDA-MB-453 breast cancer cells, but that MDA-MB-157 cells are resistant to it. Upon closer examination, we found that MDA-MB-157 cells display a high content of intracellular vesicles and cellular protrusions, compared to MDA-MB-231 cells, that could protect them from NAF-144–67. Inhibiting the formation of intracellular vesicles and dynamics of cellular protrusions of MDA-MB-157 cells, using a protein translation inhibitor (the antibiotic Cycloheximide), rendered these cells highly susceptible to NAF-144–67, suggesting that under certain conditions, the killing effect of CPPs could be augmented when they are applied in combination with an antibiotic or chemotherapy agent. These findings could prove important for the treatment of metastatic cancers with CPPs and/or treatment combinations that include CPPs.

Under natural conditions, abiotic stresses that limit plant growth and development tend to occur simultaneously, rather than individually. Due to global warming and climate change, the frequency and intensity of heat and salt stresses are becoming more frequent. Our aim is to determine the response mechanisms of tomato to different intensities of combined heat and salt stresses. The physiological and morphological responses and photosynthesis/reactive oxygen species (ROS)-related genes of tomato plants were compared under a control, heat stress, salt stress (50/100/200/400 mM NaCl), and a combination of salt and heat stresses. The stomatal conductance (gs) of tomato leaves significantly increased at a heat + 50 mM NaCl treatment on day 4, but significantly decreased at heat + 100/200/400 mM NaCl treatments, compared with the control on days 4 and 8. The O2·− production rate of tomato plants was significantly higher at heat + 100/200/400 mM NaCl than the control, which showed no significant difference between heat + 50 mM NaCl treatment and the control on days 4 and 8. Ascorbate peroxidase 2 was significantly upregulated by heat + 100/200/400 mM NaCl treatment as compared with heat + 50 mM NaCl treatment on days 4 and 8. This study demonstrated that the dominant effect ratio of combined heat and salt stress on tomato plants can shift from heat to salt, when the intensity of salt stress increased from 50 mM to 100 mM or above. This study provides important information for tomato tolerance improvement at combined heat and salt stresses.

With global climate change, the frequency and intensity of waterlogging events are increasing due to frequent and heavy precipitation. Little is known however about the response of plants to repeated waterlogging stress events. The aim is to clarify physiological regulation mechanisms of tomato plants under repeated waterlogging stress, and whether Trichoderma harzianum can alleviate waterlogging injury. We identified two genotypes of tomato, ‘MIX-002’ and ‘LA4440’, as waterlogging tolerant and sensitive genotypes, respectively, based on plant biomass accumulation. The two tomato genotypes were subjected to a waterlogging priming treatment for 2 days (excess water for 1 cm above substrate surface) followed by a recovery stage for 2 days, and then a second waterlogging stress for 5 days (excess water for 1 cm above substrate surface) followed by a second recovery stage for 3 days. Leaf physiological, plant growth parameters, and the expression of five key genes were investigated. We found that the two genotypes responded differently to waterlogging priming and stress in terms of photosynthesis, reactive oxygen species (ROS), and osmotic regulatory mechanisms. Waterlogging stress significantly increased H2O2 content of ‘MIX-002’, while that of ‘LA4440’ had no significant change. Under waterlogging stress, photosynthesis of the two genotypes treated with waterlogging priming returned to the control level. However, Trichoderma harzianum treatment during the second recovery stage did not show positive mitigative effects. The plants of ‘LA4440’ with priming showed lower peroxidase (POD) activity and proline content but higher H2O2 content than that without priming under waterlogging stress. Under waterlogging stress with priming as compared to without priming, SODCC2 was downregulated in two tomatoes, and AGR2 and X92888 were upregulated in ‘MIX-002’ but downregulated in ‘LA4440’. Overall, the two tomato genotypes exhibited distinct photosynthetic, ROS and osmotic regulatory mechanisms responding to the waterlogging stress. Waterlogging priming can induce stress memory by adjusting stomatal conductance, sustaining ROS homeostasis, regulating osmotic regulatory substances and key gene expressions mediated by H2O2, and thus alleviate the damage on tomato photosynthesis when waterlogging reoccurred.

The utmost goal of selecting an RNA-Seq alignment software is to perform accurate alignments with a robust algorithm, which is capable of detecting the various intricacies underlying read-mapping procedures and beyond. Most alignment software tools are typically pre-tuned with human or prokaryotic data, and therefore may not be suitable for applications to other organisms, such as plants. The rapidly growing plant RNA-Seq databases call for the assessment of the alignment tools on curated plant data, which will aid the calibration of these tools for applications to plant transcriptomic data. We therefore focused here on benchmarking RNA-Seq read alignment tools, using simulated data derived from the model organism Arabidopsis thaliana. We assessed the performance of five popular RNA-Seq alignment tools that are currently available, based on their usage (citation count). By introducing annotated single nucleotide polymorphisms (SNPs) from The Arabidopsis Information Resource (TAIR), we recorded alignment accuracy at both base-level and junction base-level resolutions for each alignment tool. In addition to assessing the performance of the alignment tools at their default settings, accuracies were also recorded by varying the values of numerous parameters, including the confidence threshold and the level of SNP introduction. The performances of the aligners were found consistent under various testing conditions at the base-level accuracy; however, the junction base-level assessment produced varying results depending upon the applied algorithm. At the read base-level assessment, the overall performance of the aligner STAR was superior to other aligners, with the overall accuracy reaching over 90% under different test conditions. On the other hand, at the junction base-level assessment, SubRead emerged as the most promising aligner, with an overall accuracy over 80% under most test conditions.

Marjault et al. report two crystal structures of a drug molecule bound to NEET proteins mNT and NAF-1 and the effects of this molecule on diabetic mice, suggesting an approach for reducing mitochondrial iron and ROS levels in diabetic patients.

A bifunctional peroxidase enzyme, 4-coumarate 3-hydroxylase (C3H/APX), provides a parallel route to the shikimate shunt pathway for the conversion of 4-coumarate to caffeate in the early steps of lignin biosynthesis. Knockdown of C3H/APX (C3H/APX-KD) expression has been shown to reduce the lignin content in Brachypodium distachyon. However, like many other lignin-modified plants, C3H/APX-KDs show unpredictable pleiotropic phenotypes, including stunted growth, delayed senescence, and reduced seed yield. A system-wide level understanding of altered biological processes in lignin-modified plants can help pinpoint the lignin-modification associated growth defects to benefit future studies aiming to negate the yield penalty. Here, a multi-omic approach was used to characterize molecular changes resulting from C3H/APX-KD associated lignin modification and negative growth phenotype in Brachypodium distachyon. Our findings demonstrate that C3H/APX knockdown in Brachypodium stems substantially alters the abundance of enzymes implicated in the phenylpropanoid biosynthetic pathway and disrupt cellular redox homeostasis. Moreover, it elicits plant defense responses associated with intracellular kinases and phytohormone-based signaling to facilitate growth-defense trade-offs. A deeper understanding along with potential targets to mitigate the pleiotropic phenotypes identified in this study could aid to increase the economic feasibility of lignocellulosic biofuel production.

Abstract Identifying genes that interact to confer a biological function to an organism is one of the main goals of functional genomics. High‐throughput technologies for assessment and quantification of genome‐wide gene expression patterns have enabled systems‐level analyses to infer pathways or networks of genes involved in different functions under many different conditions. Here, we leveraged the publicly available, information‐rich RNA‐Seq datasets of the model plant Arabidopsis thaliana to construct a gene co‐expression network, which was partitioned into clusters or modules that harbor genes correlated by expression. Gene ontology and pathway enrichment analyses were performed to assess functional terms and pathways that were enriched within the different gene modules. By interrogating the co‐expression network for genes in different modules that associate with a gene of interest, diverse functional roles of the gene can be deciphered. By mapping genes differentially expressing under a certain condition in Arabidopsis onto the co‐expression network, we demonstrate the ability of the network to uncover novel genes that are likely transcriptionally active but prone to be missed by standard statistical approaches due to their falling outside of the confidence zone of detection. To our knowledge, this is the first A. thaliana co‐expression network constructed using the entire mRNA‐Seq datasets (>20,000) available at the NCBI SRA database. The developed network can serve as a useful resource for the Arabidopsis research community to interrogate specific genes of interest within the network, retrieve the respective interactomes, decipher gene modules that are transcriptionally altered under certain condition or stage, and gain understanding of gene functions.

Background Therapy with mesenchymal stem cells remains a promising but challenging approach to critical limb ischemia in diabetes because of the dismal cell survival. Methods and Results Critical limb ischemia in type 2 diabetes mouse model was used to explore the impact of diabetic limb ischemia on the survival of bone marrow mesenchymal stromal cells (bMSCs). Inhibition of intracellular reactive oxygen species was achieved with concomitant overexpression of superoxide dismutase (SOD)‐1 and glutathione peroxidase‐1 in the transplanted bMSCs, and extracellular reactive oxygen species was attenuated using SOD‐3 overexpression and N‐acetylcysteine treatment. In vivo optical fluorescence imaging and laser Doppler perfusion imaging were used to track cell retention and determine blood flow in diabetic ischemic limb, respectively. Survival of the transplanted bMSCs was significantly decreased in diabetic ischemic limb compared with the control. In vitro study indicated that advanced glycation end products, not high glucose, significantly decreased the proliferation of bMSCs and increased their apoptosis associated with increased reactive oxygen species production and selective reduction of SOD‐1 and SOD‐3. In vivo study demonstrated that concomitant overexpression of SOD‐1, SOD‐3, and glutathione peroxidase‐1, or host treatment with N‐acetylcysteine, significantly enhanced in vivo survival of transplanted bMSCs, and improved critical limb ischemia in diabetic mice. Combination of triple antioxidant enzyme overexpression in bMSCs with host N‐acetylcysteine treatment further improved bMSC survival with enhanced circulatory and functional recovery from diabetic critical limb ischemia. Conclusions Simultaneous suppression of reactive oxygen species from transplanted bMSCs and host tissue could additively enhance bMSC survival in diabetic ischemic limb with increased therapeutic efficacy in diabetes.

Lignin biosynthesis in higher plants relies upon a 3-hydroxylation reaction that can occur via shikimate esters of 4-coumarate. Here, Barros et al. define an alternative biosynthetic pathway via cytosolic ascorbate peroxidase that can catalyze direct 3-hydroxylation of 4-coumarate.

NEET proteins are conserved 2Fe-2S proteins that regulate the levels of iron and reactive oxygen species in plant and mammalian cells. Previous studies of seedlings with constitutive expression of AtNEET, or its dominant-negative variant H89C (impaired in 2Fe-2S cluster transfer), revealed that disrupting AtNEET function causes oxidative stress, chloroplast iron overload, activation of iron-deficiency responses, and cell death. Because disrupting AtNEET function is deleterious to plants, we developed an inducible expression system to study AtNEET function in mature plants using a time-course proteomics approach. Here, we report that the suppression of AtNEET cluster transfer function results in drastic changes in the expression of different members of the ferredoxin (Fd), Fd-thioredoxin (TRX) reductase (FTR), and TRX network of Arabidopsis, as well as in cytosolic cluster assembly proteins. In addition, the expression of Yellow Stripe-Like 6 (YSL6), involved in iron export from chloroplasts was elevated. Taken together, our findings reveal new roles for AtNEET in supporting the Fd-TFR-TRX network of plants, iron mobilization from the chloroplast, and cytosolic 2Fe-2S cluster assembly. In addition, we show that the AtNEET function is linked to the expression of glutathione peroxidases (GPXs), which play a key role in the regulation of ferroptosis and redox balance in different organisms.

Reproductive organs and developing tissues have high energy demands that require metabolic functions primarily supported by mitochondria function. The highly conserved CISD/NEET iron-sulfur (Fe-S) protein family regulates iron and reactive oxygen homeostasis, both of which are important for mitochondrial function. Disruption of iron and reactive oxygen homeostasis typically leads to detrimental effects. In humans, CISD dysfunction is associated with human health issues including Wolfram syndrome 2. Using C. elegans, we previously determined that the cisd-1, cisd-3.1 and cisd-3.2 have an overlapping role in the regulation of physiological germline apoptosis through the canonical programmed cell death pathway. Here, we isolated the cisd-3.2(pnIs68) mutant that resulted in physiological and fitness defects including germline abnormalities that are associated with abnormal stem cell niche and disrupted formation of bivalent chromosomes. The cisd-3.2(pnIs68) mutation led to complete disruption of the cisd-3.2 gene expression and a decrease in expression of genetically intact cisd-1 and cisd-3.1 genes suggesting an indirect impact of the cisd-3.2(pnIs68) allele. The CISD-3.2 and CISD-3.1 proteins localize to the mitochondria in many tissues throughout development. The cisd-3.2(pnIs68) mutant displays phenotypes associated with mitochondrial dysfunction, including disruption of the mitochondrial network within the germline. These results further support the idea that the CISD protein family is required for mitochondrial function that supports important functions in animals including overall fitness and germline viability.

The development of plant varieties with a better nitrogen use efficiency (NUE) is a means for modern agriculture to decrease environmental pollution due to an excess of nitrate and to maintain a sufficient net income. However, the optimum environmental conditions for agriculture will tend to be more adverse in the coming years, with increases in temperatures, water scarcity, and salinity being the most important productivity constrains for plants. NUE is inherently a complex trait, as each step, including N uptake, translocation, assimilation, and remobilization, is governed by multiple interacting genetic and environmental factors. In this study, two recombinant inbred lines (RIL-66 and RIL-76) from a cross between Solanum lycopersicum and Solanum pimpinellifoilum with different degree of tolerance to the combination of salinity and heat were subjected to a physiological, ionomic, amino acid profile, and gene expression study to better understand how nitrogen metabolism is affected in tolerant plants as compared to sensitive ones. The ionomics results showed a different profile between the two RILs, with K+ and Mg2+ being significantly lower in RIL-66 (low tolerant) as compared to RIL-76 (high tolerant) under salinity and heat combination. No differences were shown between the two RILs in N total content; however, N-NO3− was significantly higher in RIL-66, whereas N-Norg was lower as compared to the other genotype, which could be correlated with its tolerance to the combination of salinity and heat. Total proteins and total amino acid concentration were significantly higher in RIL-76 as compared to the sensitive recombinant line under these conditions. Glutamate, but more importantly glutamine, was also highly synthesized and accumulated in RIL-76 under the combination of salinity and heat, which was in agreement with the upregulation of the nitrogen metabolism related transcripts studied (SlNR, SlNiR, SlGDH, SlGLT1, SlNRT1.2, SlAMT1, and SlAMT2). This study emphasized the importance of studying abiotic stress in combination and how recombinant material with different degrees of tolerance can be highly important for the improvement of nitrogen use efficiency in horticultural plants through the targeting of N-related markers.

Decreased insulin secretion, associated with pancreatic β-cell failure, plays a critical role in many human diseases including diabetes, obesity, and cancer. While numerous studies linked β-cell failure with enhanced levels of reactive oxygen species (ROS), the development of diabetes associated with hereditary conditions that result in iron overload, e.g., hemochromatosis, Friedreich’s ataxia, and Wolfram syndrome type 2 (WFS-T2; a mutation in CISD2, encoding the [2Fe-2S] protein NAF-1), underscores an additional link between iron metabolism and β-cell failure. Here, using NAF-1-repressed INS-1E pancreatic cells, we observed that NAF-1 repression inhibited insulin secretion, as well as impaired mitochondrial and ER structure and function. Importantly, we found that a combined treatment with the cell permeant iron chelator deferiprone and the glutathione precursor N-acetyl cysteine promoted the structural repair of mitochondria and ER, decreased mitochondrial labile iron and ROS levels, and restored glucose-stimulated insulin secretion. Additionally, treatment with the ferroptosis inhibitor ferrostatin-1 decreased cellular ROS formation and improved cellular growth of NAF-1 repressed pancreatic cells. Our findings reveal that suppressed expression of NAF-1 is associated with the development of ferroptosis-like features in pancreatic cells, and that reducing the levels of mitochondrial iron and ROS levels could be used as a therapeutic avenue for WFS-T2 patients.

Because of their sessile nature, plants evolved integrated defense and acclimation mechanisms to simultaneously cope with adverse biotic and abiotic conditions. Among these are systemic acquired resistance (SAR) and systemic acquired acclimation (SAA). Growing evidence suggests that SAR and SAA activate similar cellular mechanisms and employ common signaling pathways for the induction of acclimatory and defense responses. It is therefore possible to consider these processes together, rather than separately, as a common systemic acquired acclimation and resistance (SAAR) mechanism. Arabidopsis thaliana flavin-dependent monooxygenase 1 (FMO1) was previously described as a regulator of plant resistance in response to pathogens as an important component of SAR. In the current study, we investigated its role in SAA, induced by a partial exposure of Arabidopsis rosette to local excess light stress. We demonstrate here that FMO1 expression is induced in leaves directly exposed to excess light stress as well as in systemic leaves remaining in low light. We also show that FMO1 is required for the systemic induction of ASCORBATE PEROXIDASE 2 (APX2) and ZINC-FINGER OF ARABIDOPSIS 10 (ZAT10) expression and spread of the reactive oxygen species (ROS) systemic signal in response to a local application of excess light treatment. Additionally, our results demonstrate that FMO1 is involved in the regulation of excess light-triggered systemic cell death, which is under control of LESION SIMULATING DISEASE 1 (LSD1). Our study indicates therefore that FMO1 plays an important role in triggering SAA response, supporting the hypothesis that SAA and SAR are tightly connected and use the same signaling pathways.

The NEET proteins mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1) are required for cancer cell proliferation and resistance to oxidative stress. NAF-1 and mNT are also implicated in a number of other human pathologies including diabetes, neurodegeneration and cardiovascular disease, as well as in development, differentiation and aging. Previous studies suggested that mNT and NAF-1 could function in the same pathway in mammalian cells, preventing the over-accumulation of iron and reactive oxygen species (ROS) in mitochondria. Nevertheless, it is unknown whether these two proteins directly interact in cells, and how they mediate their function. Here we demonstrate, using yeast two-hybrid, in vivo bimolecular fluorescence complementation (BiFC), direct coupling analysis (DCA), RNA-sequencing, ROS and iron imaging, and single and double shRNA lines with suppressed mNT, NAF-1 and mNT/NAF-1 expression, that mNT and NAF-1 directly interact in mammalian cells and could function in the same cellular pathway. We further show using an in vitro cluster transfer assay that mNT can transfer its clusters to NAF-1. Our study highlights the possibility that mNT and NAF-1 function as part of an iron-sulfur (2Fe-2S) cluster relay to maintain the levels of iron and Fe-S clusters under control in the mitochondria of mammalian cells, thereby preventing the activation of apoptosis and/or autophagy and supporting cellular proliferation.

Lesion Simulating Disease 1 (LSD1), Enhanced Disease Susceptibility (EDS1) and Phytoalexin Deficient 4 (PAD4) were discovered a quarter century ago as regulators of programmed cell death and biotic stress responses in Arabidopsis thaliana. Recent studies have demonstrated that these proteins are also required for acclimation responses to various abiotic stresses, such as high light, UV radiation, drought and cold, and that their function is mediated through secondary messengers, such as salicylic acid (SA), reactive oxygen species (ROS), ethylene (ET) and other signaling molecules. Furthermore, LSD1, EDS1 and PAD4 were recently shown to be involved in the modification of cell walls, and the regulation of seed yield, biomass production and water use efficiency. The function of these proteins was not only demonstrated in model plants, such as Arabidopsis thaliana or Nicotiana benthamiana, but also in the woody plant Populus tremula x tremuloides. In addition, orthologs of LSD1, EDS1, and PAD4 were found in other plant species, including different crop species. In this review, we focus on specific LSD1, EDS1 and PAD4 features that make them potentially important for agricultural and industrial use.

Abiotic stresses such as drought, heat or salinity are a major cause of yield loss worldwide. Recent studies revealed that the acclimation of plants to a combination of different environmental stresses is unique and cannot be directly deduced from studying the response of plants to each of the different stresses applied individually. Here we report on the response of Arabidopsis thaliana to a combination of salt and heat stress using transcriptome analysis, physiological measurements and mutants deficient in abscisic acid, salicylic acid, jasmonic acid or ethylene signaling. Arabidopsis plants were found to be more susceptible to a combination of salt and heat stress compared to each of the different stresses applied individually. The stress combination resulted in a higher ratio of Na+/K+ in leaves and caused the enhanced expression of 699 transcripts unique to the stress combination. Interestingly, many of the transcripts that specifically accumulated in plants in response to the salt and heat stress combination were associated with the plant hormone abscisic acid. In accordance with this finding, mutants deficient in abscisic acid metabolism and signaling were found to be more susceptible to a combination of salt and heat stress than wild type plants. Our study highlights the important role abscisic acid plays in the acclimation of plants to a combination of two different abiotic stresses.

Abiotic stresses such as drought, heat or salinity are major causes of yield loss worldwide. Recent studies have revealed that the acclimation of plants to a combination of different environmental stresses is unique and therefore cannot be directly deduced from studying the response of plants to each of the different stresses applied individually. The efficient detoxification of reactive oxygen species (ROS) is thought to play a key role in enhancing the tolerance of plants to abiotic stresses. Here, we report on the role of melatonin in the protection of the photosynthetic apparatus through the increase in ROS detoxification in tomato plants grown under the combination of salinity and heat, two of the most common abiotic stresses known to act jointly. Plants treated with exogenous melatonin showed a different modulation in the expression on some antioxidant-related genes and their related enzymes. More specifically, ascorbate peroxidase, glutathione reductase, glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase (APX, GR, GPX and Ph-GPX, resepctively) showed an antagonistic regulation as compared to plants that did not receive melatonin. This translated into a better antioxidant capacity and to a lesser ROS accumulation under stress combination. The performance of the photosynthesis parameters and the photosystems was also increased in plants treated with exogenous melatonin under the combination of salinity and heat. In accordance with these findings, tomato plants treated with melatonin were found to grow better under stress combination that the non-treated ones. Our study highlights the important role that exogenous melatonin plays in the acclimation of plants to a combination of two different abiotic stresses, and how this compound can specifically regulate oxidative stress-related genes and enzymes to increase plant tolerance.

Iron-sulfur cluster biogenesis is executed by distinct protein assembly systems. Mammals have two systems, the mitochondrial Fe-S cluster assembly system (ISC) and the cytosolic assembly system (CIA), that are connected by an unknown mechanism. The human members of the NEET family of 2Fe-2S proteins, nutrient-deprivation autophagy factor-1 (NAF-1) and mitoNEET (mNT), are located at the interface between the mitochondria and the cytosol. These proteins have been implicated in cancer cell proliferation, and they can transfer their 2Fe-2S clusters to a standard apo-acceptor protein. Here we report the first physiological 2Fe-2S cluster acceptor for both NEET proteins as human Anamorsin (also known as cytokine induced apoptosis inhibitor-1; CIAPIN-1). Anamorsin is an electron transfer protein containing two iron-sulfur cluster-binding sites that is required for cytosolic Fe-S cluster assembly. We show, using UV-Vis spectroscopy, that both NAF-1 and mNT can transfer their 2Fe-2S clusters to apo-Anamorsin with second order rate constants similar to those of other known human 2Fe-2S transfer proteins. A direct protein-protein interaction of the NEET proteins with apo-Anamorsin was detected using biolayer interferometry. Furthermore, electrospray mass spectrometry of holo-Anamorsin prepared by cluster transfer shows that it receives both of its 2Fe-2S clusters from the NEETs. We propose that mNT and NAF-1 can provide parallel routes connecting the mitochondrial ISC system and the CIA. 2Fe-2S clusters assembled in the mitochondria are received by NEET proteins and when needed transferred to Anamorsin, activating the CIA.

Nutrient-deprivation autophagy factor-1 (NAF-1) (synonyms: Cisd2, Eris, Miner1, and Noxp70) is a [2Fe-2S] cluster protein immune-detected both in endoplasmic reticulum (ER) and mitochondrial outer membrane. It was implicated in human pathology (Wolfram Syndrome 2) and in BCL-2 mediated antagonization of Beclin 1-dependent autophagy and depression of ER calcium stores. To gain insights about NAF-1 functions, we investigated the biochemical properties of its 2Fe-2S cluster and sensitivity of those properties to small molecules. The structure of the soluble domain of NAF-1 shows that it forms a homodimer with each protomer containing a [2Fe-2S] cluster bound by 3 Cys and one His. NAF-1 has shown the unusual abilities to transfer its 2Fe-2S cluster to an apo-acceptor protein (followed in vitro by spectrophotometry and by native PAGE electrophoresis) and to transfer iron to intact mitochondria in cell models (monitored by fluorescence imaging with iron fluorescent sensors targeted to mitochondria). Importantly, the drug pioglitazone abrogates NAF-1's ability to transfer the cluster to acceptor proteins and iron to mitochondria. Similar effects were found for the anti-diabetes and longevity-promoting antioxidant resveratrol. These results reveal NAF-1 as a previously unidentified cell target of anti-diabetes thiazolidinedione drugs like pioglitazone and of the natural product resveratrol, both of which interact with the protein and stabilize its labile [2Fe-2S] cluster.

The concentrations of mineral nutrients in seeds are critical to both the life cycle of plants as well as human nutrition. These concentrations are strongly influenced by soil conditions, as shown here by quantifying the concentration of 14 elements in seeds from Arabidopsis thaliana plants grown under four different soil conditions: standard, or modified with NaCl, heavy metals, or alkali. Each of the modified soils resulted in a unique change to the seed ionome (the mineral nutrient content of the seeds). To help identify the genetic networks regulating the seed ionome, changes in elemental concentrations were evaluated using mutants corresponding to 760 genes as well as 10 naturally occurring accessions. The frequency of ionomic phenotypes supports an estimate that as much as 11% of the A. thaliana genome encodes proteins of functional relevance to ion homeostasis in seeds. A subset of mutants were analyzed with two independent alleles, providing five examples of genes important for regulation of the seed ionome: SOS2, ABH1, CCC, At3g14280 and CNGC2. In a comparison of nine different accessions to a Col-0 reference, eight accessions were observed to have reproducible differences in elemental concentrations, seven of which were dependent on specific soil conditions. These results indicate that the A. thaliana seed ionome is distinct from the vegetative ionome, and that elemental analysis is a sensitive approach to identify genes controlling ion homeostasis, including those that regulate gene expression, phospho-regulation, and ion transport.