RESUMO
Copper (Cu) and iron (Fe) are essential micronutrients that are toxic when accumulating in excess in cells. Thus, their uptake by roots is tightly regulated. While plants sense and respond to local Cu availability, the systemic regulation of Cu uptake has not been documented in contrast to local and systemic control of Fe uptake. Fe abundance in the phloem has been suggested to act systemically, regulating the expression of Fe uptake genes in roots. Consistently, shoot-to-root Fe signaling is disrupted in Arabidopsis thaliana mutants lacking the phloem companion cell-localized Fe transporter, OLIGOPEPTIDE TRANSPORTER 3 (AtOPT3). We report that AtOPT3 also transports Cu in heterologous systems and contributes to its delivery from sources to sinks in planta. The opt3 mutant contained less Cu in the phloem, was sensitive to Cu deficiency and mounted a transcriptional Cu deficiency response in roots and young leaves. Feeding the opt3 mutant and Cu- or Fe-deficient wild-type seedlings with Cu or Fe via the phloem in leaves downregulated the expression of both Cu- and Fe-deficiency marker genes in roots. These data suggest the existence of shoot-to-root Cu signaling, highlight the complexity of Cu/Fe interactions, and the role of AtOPT3 in fine-tuning root transcriptional responses to the plant Cu and Fe needs.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Cobre , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Floema/genética , Floema/metabolismo , Homeostase , Ferro/metabolismo , Plantas/metabolismo , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Plant responses to environmental change are mediated via changes in cellular metabolomes. However, <5% of signals obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) can be identified, limiting our understanding of how metabolomes change under biotic/abiotic stress. To address this challenge, we performed untargeted LC-MS/MS of leaves, roots, and other organs of Brachypodium distachyon (Poaceae) under 17 organ-condition combinations, including copper deficiency, heat stress, low phosphate, and arbuscular mycorrhizal symbiosis. We found that both leaf and root metabolomes were significantly affected by the growth medium. Leaf metabolomes were more diverse than root metabolomes, but the latter were more specialized and more responsive to environmental change. We found that 1 week of copper deficiency shielded the root, but not the leaf metabolome, from perturbation due to heat stress. Machine learning (ML)-based analysis annotated approximately 81% of the fragmented peaks versus approximately 6% using spectral matches alone. We performed one of the most extensive validations of ML-based peak annotations in plants using thousands of authentic standards, and analyzed approximately 37% of the annotated peaks based on these assessments. Analyzing responsiveness of each predicted metabolite class to environmental change revealed significant perturbations of glycerophospholipids, sphingolipids, and flavonoids. Co-accumulation analysis further identified condition-specific biomarkers. To make these results accessible, we developed a visualization platform on the Bio-Analytic Resource for Plant Biology website (https://bar.utoronto.ca/efp_brachypodium_metabolites/cgi-bin/efpWeb.cgi), where perturbed metabolite classes can be readily visualized. Overall, our study illustrates how emerging chemoinformatic methods can be applied to reveal novel insights into the dynamic plant metabolome and stress adaptation.
Assuntos
Brachypodium , Brachypodium/metabolismo , Cromatografia Líquida , Teoria da Informação , Cobre/metabolismo , Espectrometria de Massas em Tandem , Metabolômica/métodos , MetabolomaRESUMO
Mitochondria and chloroplasts are organelles with high iron demand that are particularly susceptible to iron-induced oxidative stress. Despite the necessity of strict iron regulation in these organelles, much remains unknown about mitochondrial and chloroplast iron transport in plants. Here, we propose that Arabidopsis ferroportin 3 (FPN3) is an iron exporter that is dual-targeted to mitochondria and chloroplasts. FPN3 is expressed in shoots, regardless of iron conditions, but its transcripts accumulate under iron deficiency in roots. fpn3 mutants cannot grow as well as the wild type under iron-deficient conditions and their shoot iron levels are lower compared with the wild type. Analyses of iron homeostasis gene expression in fpn3 mutants and inductively coupled plasma mass spectrometry (ICP-MS) measurements show that iron levels in the mitochondria and chloroplasts are increased relative to the wild type, consistent with the proposed role of FPN3 as a mitochondrial/plastid iron exporter. In iron-deficient fpn3 mutants, abnormal mitochondrial ultrastructure was observed, whereas chloroplast ultrastructure was not affected, implying that FPN3 plays a critical role in the mitochondria. Overall, our study suggests that FPN3 is essential for optimal iron homeostasis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ferro/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Cloroplastos/metabolismo , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Homeostase , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mutação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Leveduras/genética , Leveduras/metabolismoRESUMO
Iron (Fe) uptake and translocation in plants are fine-tuned by complex mechanisms that are not yet fully understood. In Arabidopsis thaliana, local regulation of Fe homeostasis at the root level has been extensively studied and is better understood than the systemic shoot-to-root regulation. While the root system is solely a sink tissue that depends on photosynthates translocated from source tissues, the shoot system is a more complex tissue, where sink and source tissues occur synchronously. In this study, and to gain better insight into the Fe deficiency responses in leaves, we overexpressed Zinc/Iron-regulated transporter-like Protein (ZIP5), an Fe/Zn transporter, in phloem-loading cells (proSUC2::AtZIP5) and determined the timing of Fe deficiency responses in sink (young leaves and roots) and source tissues (leaves). Transgenic lines overexpressing ZIP5 in companion cells displayed increased sensitivity to Fe deficiency in root growth assays. Moreover, young leaves and roots (sink tissues) displayed either delayed or dampened transcriptional responses to Fe deficiency compared to wild-type (WT) plants. We also took advantage of the Arabidopsis mutant nas4x-1 to explore Fe transcriptional responses in the opposite scenario, where Fe is retained in the vasculature but in an unavailable and precipitated form. In contrast to proSUC2::AtZIP5 plants, nas4x-1 young leaves and roots displayed a robust and constitutive Fe deficiency response, while mature leaves showed a delayed and dampened Fe deficiency response compared to WT plants. Altogether, our data provide evidence suggesting that Fe sensing within leaves can also occur locally in a leaf-specific manner.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Deficiências de Ferro , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ferro/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismoRESUMO
Age-dependent changes in reactive oxygen species (ROS) levels are critical in leaf senescence. While H2O2-reducing enzymes such as catalases and cytosolic ASCORBATE PEROXIDASE1 (APX1) tightly control the oxidative load during senescence, their regulation and function are not specific to senescence. Previously, we identified the role of ASCORBATE PEROXIDASE6 (APX6) during seed maturation in Arabidopsis (Arabidopsis thaliana). Here, we show that APX6 is a bona fide senescence-associated gene. APX6 expression is specifically induced in aging leaves and in response to senescence-promoting stimuli such as abscisic acid (ABA), extended darkness, and osmotic stress. apx6 mutants showed early developmental senescence and increased sensitivity to dark stress. Reduced APX activity, increased H2O2 level, and altered redox state of the ascorbate pool in mature pre-senescing green leaves of the apx6 mutants correlated with the early onset of senescence. Using transient expression assays in Nicotiana benthamiana leaves, we unraveled the age-dependent post-transcriptional regulation of APX6. We then identified the coding sequence of APX6 as a potential target of miR398, which is a key regulator of copper redistribution. Furthermore, we showed that mutants of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), the master regulator of copper homeostasis and miR398 expression, have a higher APX6 level compared with the wild type, which further increased under copper deficiency. Our study suggests that APX6 is a modulator of ROS/redox homeostasis and signaling in aging leaves that plays an important role in developmental- and stress-induced senescence programs.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ascorbato Peroxidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Ascorbato Peroxidases/genética , Cobre/deficiência , Proteínas de Ligação a DNA/genética , Escuridão , Homeostase , Peróxido de Hidrogênio/metabolismo , MicroRNAs/genética , Oxirredução , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Nicotiana/enzimologia , Nicotiana/genética , Nicotiana/fisiologia , Fatores de Transcrição/genéticaRESUMO
Addressing the looming global food security crisis requires the development of high-yielding crops. In agricultural soils, deficiency in the micronutrient copper significantly decreases grain yield in wheat (Triticum aestivum), a globally important crop. In cereals, grain yield is determined by inflorescence architecture, flower fertility, grain size, and weight. Whether copper is involved in these processes, and how it is delivered to the reproductive organs is not well understood. We show that copper deficiency alters not only the grain set but also flower development in both wheat and its recognized model, Brachypodium distachyon. We then show that the Brachypodium yellow stripe-like 3 (YSL3) transporter localizes to the phloem, transports copper in frog (Xenopus laevis) oocytes, and facilitates copper delivery to reproductive organs and grains. Failure to deliver copper, but not iron, zinc, or manganese to these structures in the ysl3 CRISPR-Cas9 mutant results in delayed flowering, altered inflorescence architecture, reduced floret fertility, grain size, weight, and protein accumulation. These defects are rescued by copper supplementation and are complemented by YSL3 cDNA. This knowledge will help to devise sustainable approaches for improving grain yield in regions where soil quality is a major obstacle for crop production. Copper distribution by a phloem-localized transporter is essential for the transition to flowering, inflorescence architecture, floret fertility, size, weight, and protein accumulation in seeds.
Assuntos
Brachypodium/fisiologia , Cobre/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , Brachypodium/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , ReproduçãoRESUMO
A deficiency of the micronutrient copper (Cu) leads to infertility and grain/seed yield reduction in plants. How Cu affects fertility, which reproductive structures require Cu, and which transcriptional networks coordinate Cu delivery to reproductive organs is poorly understood. Using RNA-seq analysis, we showed that the expression of a gene encoding a novel transcription factor, CITF1 (Cu-DEFICIENCY INDUCED TRANSCRIPTION FACTOR1), was strongly upregulated in Arabidopsis thaliana flowers subjected to Cu deficiency. We demonstrated that CITF1 regulates Cu uptake into roots and delivery to flowers and is required for normal plant growth under Cu deficiency. CITF1 acts together with a master regulator of copper homeostasis, SPL7 (SQUAMOSA PROMOTER BINDING PROTEIN LIKE7), and the function of both is required for Cu delivery to anthers and pollen fertility. We also found that Cu deficiency upregulates the expression of jasmonic acid (JA) biosynthetic genes in flowers and increases endogenous JA accumulation in leaves. These effects are controlled in part by CITF1 and SPL7. Finally, we show that JA regulates CITF1 expression and that the JA biosynthetic mutant lacking the CITF1- and SPL7-regulated genes, LOX3 and LOX4, is sensitive to Cu deficiency. Together, our data show that CITF1 and SPL7 regulate Cu uptake and delivery to anthers, thereby influencing fertility, and highlight the relationship between Cu homeostasis, CITF1, SPL7, and the JA metabolic pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Cobre/farmacologia , Ciclopentanos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fertilidade/fisiologia , Oxilipinas/metabolismo , Pólen/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Vias Biossintéticas/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cobre/deficiência , Ciclopentanos/farmacologia , Proteínas de Ligação a DNA/genética , Fertilidade/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Homeostase , Modelos Biológicos , Mutação/genética , Oxilipinas/farmacologia , Fenótipo , Pólen/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The mechanisms of root iron uptake and the transcriptional networks that control root-level regulation of iron uptake have been well studied, but the mechanisms by which shoots signal iron status to the roots remain opaque. Here, we characterize an Arabidopsis (Arabidopsis thaliana) double mutant, yellow stripe1-like yellow stripe3-like (ysl1ysl3), which has lost the ability to properly regulate iron deficiency-influenced gene expression in both roots and shoots. In spite of markedly low tissue levels of iron, the double mutant does not up- and down-regulate iron deficiency-induced and -repressed genes. We have used grafting experiments to show that wild-type roots grafted to ysl1ysl3 shoots do not initiate iron deficiency-induced gene expression, indicating that the ysl1ysl3 shoots fail to send an appropriate long-distance signal of shoot iron status to the roots. We present a model to explain how impaired iron localization in leaf veins results in incorrect signals of iron sufficiency being sent to roots and affecting gene expression there. Improved understanding of the mechanism of long-distance iron signaling will allow improved strategies for the engineering of staple crops to accumulate additional bioavailable iron in edible parts, thus improving the iron nutrition of the billions of people worldwide whose inadequate diet causes iron deficiency anemia.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácido Azetidinocarboxílico/análogos & derivados , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transdução de Sinais , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ácido Azetidinocarboxílico/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucuronidase/metabolismo , Ferro/farmacologia , Modelos Biológicos , Mutação/genética , Floema/metabolismo , Exsudatos de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plântula/efeitos dos fármacos , Plântula/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria por Raios XRESUMO
Iron is essential for both plant growth and human health and nutrition. Knowledge of the signaling mechanisms that communicate iron demand from shoots to roots to regulate iron uptake as well as the transport systems mediating iron partitioning into edible plant tissues is critical for the development of crop biofortification strategies. Here, we report that OPT3, previously classified as an oligopeptide transporter, is a plasma membrane transporter capable of transporting transition ions in vitro. Studies in Arabidopsis thaliana show that OPT3 loads iron into the phloem, facilitates iron recirculation from the xylem to the phloem, and regulates both shoot-to-root iron signaling and iron redistribution from mature to developing tissues. We also uncovered an aspect of crosstalk between iron homeostasis and cadmium partitioning that is mediated by OPT3. Together, these discoveries provide promising avenues for targeted strategies directed at increasing iron while decreasing cadmium density in the edible portions of crops and improving agricultural productivity in iron deficient soils.
RESUMO
Urea is an important source of nitrogen (N) for the growth and development of plants. It occurs naturally in soils, is the major N source in agricultural fertilizers and is an important N metabolite in plants. Therefore, the identification and characterization of urea transporters in higher plants is important for the fundamental understanding of urea-based N nutrition in plants and for designing novel strategies for improving the N-use efficiency of urea based-fertilizers. Progress in this area, however, is hampered due to scarce knowledge of plant urea transporters. From what is known, urea uptake from the soil into plant roots is mediated by two types of transporters: the major intrinsic proteins (MIPs) and the DUR3 orthologs, mediating low- and high-affinity urea transport, respectively. Here we characterized a MIP family member from Cucumis sativus, CsNIP2;1, with regard to its contribution to urea transport. We show that CsNIP2;1 is a plasma membrane transporter that mediates pH-dependent urea uptake when expressed in yeast. We also found that ectopic expression of CsNIP2;1 improves growth of wild-type Arabidopsis thaliana and rescues growth and development of the atdur3-3 mutant on medium with urea as the sole N source. In addition, CsNIP2;1 is transcriptionally up-regulated by N deficiency, urea and NO3 (-). These data and results from the analyses of the pattern of CsNIP2;1 expression in A. thaliana and cucumber suggest that CsNIP2;1 might be involved in multiple steps of urea-based N nutrition, including urea uptake and internal transport during N remobilization throughout seed germination and N delivery to developing tissues.
Assuntos
Arabidopsis/genética , Membrana Celular/metabolismo , Cucumis sativus/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/metabolismo , Ureia/metabolismo , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Cucumis sativus/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Teste de Complementação Genética , Glicerol/farmacologia , Proteínas de Membrana Transportadoras/química , Mutação/genética , Nitratos/metabolismo , Nitrogênio/farmacologia , Proteínas de Plantas/química , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transporte Proteico/efeitos dos fármacos , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Transformação Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Among the mechanisms controlling copper homeostasis in plants is the regulation of its uptake and tissue partitioning. Here we characterized a newly identified member of the conserved CTR/COPT family of copper transporters in Arabidopsis thaliana, COPT6. We showed that COPT6 resides at the plasma membrane and mediates copper accumulation when expressed in the Saccharomyces cerevisiae copper uptake mutant. Although the primary sequence of COPT6 contains the family conserved domains, including methionine-rich motifs in the extracellular N-terminal domain and a second transmembrane helix (TM2), it is different from the founding family member, S. cerevisiae Ctr1p. This conclusion was based on the finding that although the positionally conserved Met(106) residue in the TM2 of COPT6 is functionally essential, the conserved Met(27) in the N-terminal domain is not. Structure-function studies revealed that the N-terminal domain is dispensable for COPT6 function in copper-replete conditions but is important under copper-limiting conditions. In addition, COPT6 interacts with itself and with its homolog, COPT1, unlike Ctr1p, which interacts only with itself. Analyses of the expression pattern showed that although COPT6 is expressed in different cell types of different plant organs, the bulk of its expression is located in the vasculature. We also show that COPT6 expression is regulated by copper availability that, in part, is controlled by a master regulator of copper homeostasis, SPL7. Finally, studies using the A. thaliana copt6-1 mutant and plants overexpressing COPT6 revealed its essential role during copper limitation and excess.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Transportador de Cobre 1 , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Homeostase , Proteínas de Membrana Transportadoras/metabolismo , Microscopia Confocal/métodos , Mutagênese Sítio-Dirigida , Plantas Geneticamente Modificadas , Proteínas SLC31 , Fatores de Transcrição/genéticaRESUMO
The bone morphogenetic protein (BMP) signaling pathway regulates multiple developmental and homeostatic processes. Mutations in the pathway can cause a variety of somatic and hereditary disorders in humans. Multiple levels of regulation, including extracellular regulation, ensure proper spatiotemporal control of BMP signaling in the right cellular context. We have identified a modulator of the BMP-like Sma/Mab pathway in C. elegans called DRAG-1. DRAG-1 is the sole member of the repulsive guidance molecule (RGM) family of proteins in C. elegans, and is crucial in regulating body size and mesoderm development. Using a combination of molecular genetic and biochemical analyses, we demonstrate that DRAG-1 is a membrane-associated protein that functions at the ligand-receptor level to modulate the Sma/Mab pathway in a cell-type-specific manner. We further show that DRAG-1 positively modulates this BMP-like pathway by using a novel Sma/Mab-responsive reporter. Our work provides a direct link between RGM proteins and BMP signaling in vivo and a simple and genetically tractable system for mechanistic studies of RGM protein regulation of BMP pathways.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Caenorhabditis elegans , Proteínas de Membrana/metabolismo , Transdução de Sinais/genética , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Humanos , Proteínas de Membrana/genética , Mutação , Neurônios/metabolismo , Ligação Proteica/genética , Proteínas/genética , Proteínas/metabolismoRESUMO
The chronic exposure of humans to the toxic metal cadmium (Cd), either occupational or from food and air, causes various diseases, including neurodegenerative conditions, dysfunction of vital organs, and cancer. While the toxicology of Cd and its effect on the homeostasis of biologically relevant elements is increasingly recognized, the spatial distribution of Cd and other elements in Cd toxicity-caused diseases is still poorly understood. Here, we use Caenorhabditis elegans as a non-mammalian multicellular model system to determine the distribution of Cd at the tissue and cellular resolution and its effect on the internal levels and the distribution of biologically relevant elements. Using inductively coupled plasma-mass spectrophotometry (ICP-MS), we show that exposure of worms to Cd not only led to its internal accumulation but also significantly altered the C. elegans ionome. Specifically, Cd treatment was associated with increased levels of toxic elements such as arsenic (As) and rubidium (Rb) and a decreased accumulation of essential elements such as zinc (Zn), copper (Cu), manganese (Mn), calcium (Ca), cobalt (Co) and, depending on the Cd-concentration used in the assay, iron (Fe). We regarded these changes as an ionomic signature of Cd toxicity in C. elegans. We also show that supplementing nematode growth medium with Zn but not Cu, rescues Cd toxicity and that mutant worms lacking Zn transporters CDF-1 or SUR-7, or both are more sensitive to Cd toxicity. Finally, using synchrotron X-Ray fluorescence Microscopy (XRF), we showed that Cd significantly alters the spatial distribution of mineral elements. The effect of Cd on the distribution of Fe was particularly striking: while Fe was evenly distributed in intestinal cells of worms grown without Cd, in the presence of Cd, Fe, and Cd co-localized in punctum-like structures in the intestinal cells. Together, this study advances our understanding of the effect of Cd on the accumulation and distribution of biologically relevant elements. Considering that C. elegans possesses the principal tissues and cell types as humans, our data may have important implications for future therapeutic developments aiming to alleviate Cd-related pathologies in humans.
RESUMO
Recent improvements in synchrotron-based X-ray fluorescence (SXRF) microscopy established it as an advanced analytical tool for analyzing 2D- and 3D distribution of mineral elements in plants. Among existing imaging techniques, SXRF microscopy offers several unique capabilities, including in situ metal quantification in plant tissues and high sensitivity, as low as 1 mg kg-1, at the nanoscale spatial resolution. SXRF is increasingly utilized in different plant science disciplines to provide a fundamental understanding of metal homeostasis, and the function of trace elements in plant metabolism and development. Here, we describe methods for SXRF imaging, including sample preparation, the optimization of conventional SXRF for analyzing trace elements, and the development of confocal SXRF (C-SXRF).
Assuntos
Oligoelementos , Raios X , Síncrotrons , Metais/metabolismo , Microscopia de Fluorescência , Plantas/metabolismo , Espectrometria por Raios X/métodosRESUMO
BACKGROUND: To balance the demand for uptake of essential elements with their potential toxicity living cells have complex regulatory mechanisms. Here, we describe a genome-wide screen to identify genes that impact the elemental composition ('ionome') of yeast Saccharomyces cerevisiae. Using inductively coupled plasma - mass spectrometry (ICP-MS) we quantify Ca, Cd, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, S and Zn in 11890 mutant strains, including 4940 haploid and 1127 diploid deletion strains, and 5798 over expression strains. RESULTS: We identified 1065 strains with an altered ionome, including 584 haploid and 35 diploid deletion strains, and 446 over expression strains. Disruption of protein metabolism or trafficking has the highest likelihood of causing large ionomic changes, with gene dosage also being important. Gene over expression produced more extreme ionomic changes, but over expression and loss of function phenotypes are generally not related. Ionomic clustering revealed the existence of only a small number of possible ionomic profiles suggesting fitness tradeoffs that constrain the ionome. Clustering also identified important roles for the mitochondria, vacuole and ESCRT pathway in regulation of the ionome. Network analysis identified hub genes such as PMR1 in Mn homeostasis, novel members of ionomic networks such as SMF3 in vacuolar retrieval of Mn, and cross-talk between the mitochondria and the vacuole. All yeast ionomic data can be searched and downloaded at http://www.ionomicshub.org. CONCLUSIONS: Here, we demonstrate the power of high-throughput ICP-MS analysis to functionally dissect the ionome on a genome-wide scale. The information this reveals has the potential to benefit both human health and agriculture.
Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Cátions/genética , Redes Reguladoras de Genes , Íons/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Bases , Perfilação da Expressão Gênica , Genoma Fúngico , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Canais Iônicos/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Família Multigênica/genética , Transportadores de Ânions Orgânicos/genética , Fenótipo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNARESUMO
MOTIVATION: High-throughput perturbation screens measure the phenotypes of thousands of biological samples under various conditions. The phenotypes measured in the screens are subject to substantial biological and technical variation. At the same time, in order to enable high throughput, it is often impossible to include a large number of replicates, and to randomize their order throughout the screens. Distinguishing true changes in the phenotype from stochastic variation in such experimental designs is extremely challenging, and requires adequate statistical methodology. RESULTS: We propose a statistical modeling framework that is based on experimental designs with at least two controls profiled throughout the experiment, and a normalization and variance estimation procedure with linear mixed-effects models. We evaluate the framework using three comprehensive screens of Saccharomyces cerevisiae, which involve 4940 single-gene knock-out haploid mutants, 1127 single-gene knock-out diploid mutants and 5798 single-gene overexpression haploid strains. We show that the proposed approach (i) can be used in conjunction with practical experimental designs; (ii) allows extensions to alternative experimental workflows; (iii) enables a sensitive discovery of biologically meaningful changes; and (iv) strongly outperforms the existing noise reduction procedures. AVAILABILITY: All experimental datasets are publicly available at www.ionomicshub.org. The R package HTSmix is available at http://www.stat.purdue.edu/~ovitek/HTSmix.html. CONTACT: ovitek@stat.purdue.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Ensaios de Triagem em Larga Escala , Fenótipo , Técnicas de Inativação de Genes , Genômica , Modelos Lineares , Saccharomyces cerevisiae/genéticaRESUMO
Phytochelatins mediate tolerance to heavy metals in plants and some fungi by sequestering phytochelatin-metal complexes into vacuoles. To date, only Schizosaccharomyces pombe Hmt1 has been described as a phytochelatin transporter and attempts to identify orthologous phytochelatin transporters in plants and other organisms have failed. Furthermore, recent data indicate that the hmt1 mutant accumulates significant phytochelatin levels in vacuoles, suggesting that unidentified phytochelatin transporters exist in fungi. Here, we show that deletion of all vacuolar ABC transporters abolishes phytochelatin accumulation in S. pombe vacuoles and abrogates (35)S-PC(2) uptake into S. pombe microsomal vesicles. Systematic analysis of the entire S. pombe ABC transporter family identified Abc2 as a full-size ABC transporter (ABCC-type) that mediates phytochelatin transport into vacuoles. The S. pombe abc1 abc2 abc3 abc4 hmt1 quintuple and abc2 hmt1 double mutant show no detectable phytochelatins in vacuoles. Abc2 expression restores phytochelatin accumulation into vacuoles and suppresses the cadmium sensitivity of the abc quintuple mutant. A novel, unexpected, function of Hmt1 in GS-conjugate transport is also shown. In contrast to Hmt1, Abc2 orthologs are widely distributed among kingdoms and are proposed as the long-sought vacuolar phytochelatin transporters in plants and other organisms.
Assuntos
Cádmio/metabolismo , Mutação , Fitoquelatinas/metabolismo , Schizosaccharomyces/enzimologia , Vacúolos/enzimologia , Transportadores de Cassetes de Ligação de ATP , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Cádmio/farmacologia , Fitoquelatinas/genética , Schizosaccharomyces/genética , Vacúolos/genéticaRESUMO
Uptake and internal transport of micronutrients are essential for plant growth, development, and yield. In this regard, Iron Regulated Transporters (IRTs) from the Zinc Regulated Transporter (ZRT)/IRT-related protein (ZIP) family play an important role in transition metal uptake. Most studies have been focused on IRT1-like proteins in diploid species. Information on IRT1-like proteins in polyploids is limited. Here, we studied the function of TpIRT1A and TpIRT1B homoeologs in a tetraploid crop, Polish wheat (Triticum polonicum L.). Our results highlighted the importance of TpIRT1 in mediating the uptake and translocation of Fe, Mn, Co, and Cd with direct implications for wheat yield potential. Both TpIRT1A and TpIRT1B were located at the plasma membrane and internal vesicle-like organelle in protoplasts of Arabidopsis thaliana L. and increased Cd and Co sensitivity in yeast. The over-expression of TpIRT1B in A. thaliana increased Fe, Mn, Co, and Cd concentration in its tissues and improved plant growth under Fe, Mn, and Co deficiencies, while increased the sensitivity to Cd compared to wild type. Functional analysis of IRT1 homoeologs from tetraploid and diploid ancestral wheat species in yeast disclosed four distinct amino acid residues in TdiIRT1B (T. dicoccum L. (Schrank)) and TtuIRT1B (T. turgidum L.). Together, our results increase the knowledge of IRT1 function in a globally important crop, wheat.