EVALUACIÓN DEL METABOLISMO DEL GLUTATIÓN EN LA TOLERANCIA A METALES PESADOS

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UNIVERSIDAD AUTÓNOMA DE MADRID FACULTAD DE CIENCIAS DEPARTAMENTO DE BIOLOGÍA EVALUACIÓN DEL METABOLISMO DEL GLUTATIÓN EN LA TOLERANCIA A METALES PESADOS JUAN SOBRINO PLATA MADRID, Memoria para optar
UNIVERSIDAD AUTÓNOMA DE MADRID FACULTAD DE CIENCIAS DEPARTAMENTO DE BIOLOGÍA EVALUACIÓN DEL METABOLISMO DEL GLUTATIÓN EN LA TOLERANCIA A METALES PESADOS JUAN SOBRINO PLATA MADRID, 2014 Memoria para optar al grado de doctor realizada en el Departamento de Biología de la Facultad de Ciencias en la Universidad Autónoma de Madrid y que lleva por título: EVALUACIÓN DEL METABOLISMO DEL GLUTATIÓN EN LA TOLERANCIA A METALES PESADOS Presentada por: Juan Sobrino Plata Licenciado en Biología y en Bioquímica. Dirigida por Luis Eduardo Hernández Rodríguez Profesor titular Dpo. Biología Universidad Autónoma de Madrid Carolina Escobar Lucas Profesor titular Dpo. Ciencias Ambientales Universidad de Castilla la Mancha Madrid, 2014 Abreviaturas APS ATPS Ala APR As ASA APX Cd CAT Cys DHAR γec γecs Glu GSH GR GS Gly hgsh MDA Hg MDHAR OAS OAS-TL GSSG POX PC PCS PAGE ROS SULTR SiR SOD 5 adenosine phosphosulfate Adenosine triphosphate sulfurylase Alanine APS reductase Arsenic Ascorbate Ascorbate peroxidase Cadmium Catalase Cysteine Dehydro ascorbate reductase gamma-glutamylcysteine gamma-glutamylcysteine synthetase Glutamate Glutathione Glutathione reductase Glutathione synthetase Glycine homo-glutathione Malondialdehyde Mercury Monodehydro ascorbate reductase O-acetylserine O-acetylserine (thiol) lyase Oxidized glutathione Peroxidase Phytochelatin Phytochelatin synthase polyacrylamide gel electrophoresis Reactive oxygen species Sulfate transporter Sulfite reductase Superoxide dismutase AGRADECIMIENTOS El desarrollo de esta tesis doctoral fue financiado por las siguientes instituciones: Junta de Comunidades de Castilla-La Mancha. Proyectos: POII (FITOALMA2) y PBI (FITOALMA). Ministerio de Economía y Competitividad. Proyecto: AGL (PROBIOMET). Comunidad de Madrid. Consorcio EIADES, proyecto: S2009/AMB XIV Programa Nacional de Investigación Científica Fundación Ramón Areces Resumen Se estudiaron las respuestas de estrés oxidativo inducido por cadmio (Cd) y mercurio (Hg), característico síntoma de toxicidad por metales, en plantas de alfalfa crecidas en un medio semi-hidropónico con diferentes dosis de metal (0, 3, 10 y 30 µm) durante una semana. Ambos metales produjeron alteraciones en enzimas antioxidantes, siendo el Hg más tóxico que el Cd, destacando la inhibición de la actividad glutatión reductasa (GR), mientras que con Cd hubo mayor síntesis de fitoquelatinas (PCs). Estás diferencias en los mecanismos de toxicidad nos llevó a plantear la posibilidad de identificar las firmas de estrés específicas para elementos tóxicos (Cd, Hg y arsénico (As); 0, 6 y 30 µm) en la planta metalofita Silene vulgaris. Tras una semana de exposición, se confirmó que la inhibición de GR era un indicador específico de estrés por Hg, siendo de nuevo importante la producción de PCs en plantas tratadas con Cd. Asimismo, el Cd produjo fotoinhibición y alteración en proteínas relacionadas con el aparato fotosintético, lo que pudo estar relacionado con la mayor translocación de este metal a hoja. La evidente implicación del GSH y otros biotioles en la respuesta a metal(loide)s tóxicos nos llevó a plantear el experimentos funcionales para caracterizar en detalle su contribución en las respuestas Cd y Hg, para lo que utilizaron mutantes de Arabidopsis thaliana con niveles alterados de GSH y biotioles frente al ecotipo silvestre (Col- 0). Se trabajó con tres alelos mutantes de la enzima γ-glutamilcisteina sintetasa (γecs), pad2-1, cad2-1, y rax1-1 (acumulan 20, 30 y 45% del GSH detectado en Col-0, respectivamente); y un mutante defectivo en fitoquelatina sintasa (PCS) cad1-3 (que no acumula PCs). Se diseñaron dos aproximaciones distintas, por un lado se infiltraron hojas de plantas Col-0, cad2-1, rax1-1 y cad1-3 o se cultivaron en un sistema hidropónico puro con Cd y Hg (0, 3, 10 o 30 µm) durante h. Se observó que pequeñas diferencias en el contenido de GSH hace que la planta se enfrente al estrés de diferente manera, teniendo comportamientos similares a Col-0 en rax1-1, mientras que pad2-1 mostró la mayor sensibilidad. También se estudió el perfil transcripcional de genes de la ruta de asimilación de azufre y metabolismo del GSH en plantas de Arabidopsis Col-0, mutante de γecs y cad1-3, tratadas con un nivel moderado de Hg (3 µm) durante 72 h. Se confirmó que los mutantes cad2-1 y pad2-1 fueron particularmente sensibles a Hg, al tiempo que es importante el nivel de GSH en el patrón de expresión génica. Nuestros datos apoyan la noción de un umbral crítico de concentración de GSH, observada en los mutantes rax1-1 con niveles de GSH más parecidos a Col-0, que permite tolerancia a Cd y Hg. Se ha puesto de manifiesto la importancia del GSH en la tolerancia, tanto a nivel del ajuste metabólico de la respuesta, como en la capacidad de absorción y transporte de metales y metaloides tóxicos, observándose respuestas específicas para cada contaminante. Esta información puede ser útil para la optimización de estrategias de descontaminación de suelos contaminados por elementos tóxicos mediante fitorremediación. Abstract We studied the responses of oxidative stress induced by cadmium (Cd) and mercury (Hg), a characteristic symptom of metal toxicity, in alfalfa plants grown on a semi-hydroponic medium with different doses of metal (0, 3, 10 and 30 µm) for a week. Both metals produced alterations in antioxidant enzymes, being Hg more toxic than Cd, in particular the strong inhibition of glutathione reductase (GR) activity, while there was greater synthesis of phytochelatins (PCs) with Cd. The observed differences in the mechanisms of toxicity led us to identifying the specific stress signatures for different toxic elements (Cd, Hg and arsenic (As); 0, 6 and 30 µm) in the metallophyte Silene vulgaris. After a week of exposure, we confirmed that GR inhibition was a specific indicator of stress by Hg, being Cd a potent inductor of PCs production. Cadmium also caused photoinhibition and alteration in proteins related to the photosynthetic apparatus, which could be related to increased translocation of this metal to the shoot. The involvement of GSH and other biothiols in response to toxic metal(loid)s prompted us to perform functional experiments to characterize in detail its contribution in the responses to Cd and Hg, using mutants of Arabidopsis thaliana with altered levels of GSH and biothiols compared with the wild type (Col-0). We studied three mutant alleles of the enzyme γ-glutamylcysteine synthetase (γecs), pad2-1, cad2-1and rax1-1 (that contain 20, 30 and 45% of Col-0 GSH levels, respectively); and a mutant defective in phytochelatin synthase (PCS) cad1-3 (does not accumulate PCs). Two different approaches were designed, on the one hand infiltrated leaves of plants Col-0, cad2-1, rax1-1 and cad1-3, or were cultivated in a pure hydroponic system with Cd and Hg (0, 3, 10 or 30 µm) for h. It was observed that small differences in the content of GSH makes the plant to face stress differently, showing similar behaviors Col-0 and rax1-1, while pad2-1 was more sensitivity. It was also studied the transcriptional profile of genes of the assimilation of sulfur pathway and GSH metabolism in Arabidopsis Col-0, the γecs mutant plants of and cad1-3, treated with a moderate level of Hg (3 µm) for 72 h. It was confirmed that the cad2-1 and pad2-1 mutants were particularly sensitive to Hg, at the time that the level of GSH is important for the gene expression profile. Our data support the notion of a critical threshold of GSH concentration, observed in the rax1-1 mutant with GSH levels more similar to Col-0, which allows tolerance to Cd and Hg. It has been highlighted the importance of GSH in tolerance, both at the level of metabolic adjustment of the response, and at the level of toxic metal and metalloids absorption and transport, with specific responses for each pollutant. This information can be useful for the optimization of the decontamination strategies of soils polluted with toxic elements using phytoremediation technologies. Índice Capítulo 1: Biothiols metabolism is crucial for plant cell tolerance to toxic metals and metalloids: the case of mercury Abstract The great challenge of environmental pollution with toxic elements Remediation strategies. Phytoremediation Damages induced by toxic elements in plants Plant tolerance and detoxification mechanisms Glutathione, the key metabolite in redox homeostasis Glutathione metabolism and metal(loid) tolerance: the case of Hg References Objetivos Capítulo 2: Differential alterations of antioxidant defenses as bioindicators of mercury and cadmium toxicity in alfalfa Abstract Introduction Materials and Methods Results Discussion Acknowledgments References Supplementary Material Capítulo 3: Specific stress responses to cadmium, arsenic and mercury appear in the metallophyte Silene vulgaris when grown hydroponically Abstract Introduction Experimental Results Discussion Conclusion Acknowledgments References Supplementary Material... 90 Capítulo 4: The role of glutathione in mercury tolerance resembles its function under cadmium stress in Arabidopsis Abstract Introduction Experimental Results Discussion Acknowledgments References Supplementary Material Capítulo 5: Glutathione is a key antioxidant metabolite to cope with mercury and cadmium stress Abstract Introduction Materials and Methods Results Discussion Conclusions Acknowledgments References Supplementary Material Capítulo 6: Characterization of sulfur and glutathione metabolism responses to mercury in glutathione defective Arabidopsis mutants Introduction Materials and Methods Results Discussion References Supplementary Material Consideraciones generales y conclusiones CAPÍTULO 1. INTRODUCCIÓN Biothiols metabolism is crucial for plant cell tolerance to toxic metals and metalloids: the case of mercury Tesis doctoral Juan Sobrino Plata Capítulo 1. Introducción: Biothiols metabolism is crucial for plant cell tolerance to toxic metals and metalloids: the case of mercury Juan Sobrino-Plata 1,2, Carolina Escobar 2, Luis E. Hernández 1 1 Laboratory of Plant Physiology, Department of Biology, Universidad Autónoma de Madrid, Cantoblanco, ES Madrid. 2 Departamento de Ciencias del Medioambiente, Universidad de Castilla-La Mancha, Campus Fábrica de Armas, ES Toledo, Spain. ABSTRACT In this review we summarize the latest findings about the involvement of biothiol metabolism in metal tolerance in plants, focusing our attention to mercury (Hg), one of the most hazardous metals to the environment. The assimilation of sulfur, the synthesis of glutathione (GSH) and the accumulation of phytochelatins (PCs) are processes of biothiol metabolism critical for tolerance to toxic elements, as they contribute to maintain the redox cellular homeostasis and limit the concentration of free metals and metalloids ions. 1. The great challenge of environmental pollution with toxic elements. 1.1 Heavy metals or toxic elements? The term heavy metal has no consensus meaning to identify an element, as there are different connotations in the literature based on its density, atomic weight, atomic number, chemical properties or toxicity (Duffus 2002). In general, heavy metals include those elements with a specific density higher than 5 g/cm 3, although frequently some metalloids such as arsenic (As) are included in this group only by means of their well-known toxicity. Among the different ways to classify heavy metals, perhaps one of the most accurate from a physiological point of view is based on their functions in living organisms. Thus, several heavy metals are classified as essential when are required for metabolic processes in the cell, such as iron (Fe), copper (Cu), cobalt (Co), manganese (Mn), magnesium (Mg) or zinc (Zn), which are mainly enzymatic cofactors, and are only toxic above a threshold concentration. On the other hand, non-essential heavy metals are toxic even at low 1 Glutatión y tolerancia a metales tóxicos concentrations, being cadmium (Cd), chromium (Cr), lead (Pb), mercury (Hg), aluminum (Al) and As the most relevant to the environment (Tchounwou et al. 2012); elements that we prefer to name as toxic elements. 1.2 Sources of toxic elements and the public concern. Important amounts of toxic elements are released by the Earth geological (volcanic or geothermal) activity and rock weathering and erosion, as these elements are found frequently in nature associated with metal sulfide ores, such as occurs with Cd together with Zn (sphalerite, ZnS), the presence of As with pyrite (FeS 2 ), chalcopyrite (CuFeS 2 ) or galena (PbS), or the release of Hg from cinnabar (HgS) (Ziemacki et al. 1989). However, the largest proportion of toxic elements accumulating in the environment comes from several human activities during centuries. Nriagu (1996) affirms that anthropogenic accumulation of metals stated with the domestication of fire and the development of metallurgy, which augmented remarkably with the beginning of the Industrial Revolution. The use of metals and metalloids in modern society and industry, with novel uses in current technology, has increased enormously. Examples of this are the spread of fertilizers and pesticides containing Cd or As in agricultural environments, the addition of lead to gasoline, manufacturing of metallic paintings and batteries, elaboration of metal containing plastics, use of Hg in lighting systems and electronics or in medicine (dental amalgams, antiseptics) (Järup, 2003). Besides these uses, mining activities during many years, the increase of exhaust fumes or the release of industrial wastes have also contaminated vast areas (Alloway 2012). It is relevant to mention that seven of the top ten sites most polluted in the World in 2013 were drastically contaminated by metals and metalloids (Blacksmith Institute and Green Cross Switzerland 2013; see Fig. 1 for details). Some examples are The Citarum River in Indonesia, which covers an area of approximately Km 2 with a population of 9 million people that consume water with concentrations of Al, Mn and Fe four-times higher than recommended levels for water consume by the EPA (United Sates Environmental Protection Agency). Another examples are the Matanza Riachuelo in Argentina, which contains great amounts of Zn, Pb, Cu, Ni and Cr released from industries in the area, or alarming Pb contamination in Kabwe (Zambia), caused by intense and uncontrolled mining activity since 1902, that is causing that children s blood Pb levels exceed 5-10 times the recommended level. 2 Tesis doctoral Juan Sobrino Plata Fig. 1 The top ten most polluted sites worldwide according to databases collected by Blacksmith Institute and Green Cross Switzerland Number of people affected in these places and causes of this contamination are cited in each case. One of the major problems of toxic elements is their persistence in the environment, as they can spread to different ecosystem compartments (water, soil), bioaccumulate and biomagnify in the trophic chain, which eventually constitutes a health risk to humans (Järup 2003). Consumption of contaminated aliments is associated with different illness or syndromes, with specific effects over human health, although most of them are considered carcinogens. It is well known that Cd promotes physiological damages in kidneys, lung, liver or bones (Järup and Åkesson 2009). The ingest of Pb and long-term exposure causes problems in the nervous system, encephalopathy, abdominal pain, chronic headache; in children can cause behavioral disturbances, difficulties in learning and concentration, even diminishing the intellectual capacity (Lidsky and Schneider 2003, Needleman 2004). Arsenic can produce severe disturbances of cardiovascular and central nervous system, gastrointestinal symptoms and long-term inhalation or ingest can produce lung, kidney, skin or bladder cancer (Kapaj et al. 2006). These examples, and many more (see below), have augmented the public concern about the uncontrolled use and release of toxic elements to the environment, and new regulatory rules and laws have been established 3 Glutatión y tolerancia a metales tóxicos recently by different international and national committees and governing institutions (EPA, EU, FAO, etc.). Therefore, limited used of some metal(loid)s, improved waste management, controlled pollutant-emissions or developing of sustainable procedures to cleanup contaminated sites are now tasks for the benefit of human health (Blacksmith Institute and Green Cross Switzerland 2013). 1.3 Mercury, the silent poison. Mercury has chemical and physical properties make it a unique element, being one of the most hazardous metal(loid)s in nature. In 1997 the EPA elaborated a report about Hg sources, risk for human health or potential control technologies, recommending the reduction of the use of Hg (Keating et al. 1997). Mercury is today a global environmental problem: the Environment Programme of United Nations (UNEP) has a special ad-hoc work group on Hg where the scientific community can contribute to the negotiations of an internationally legal instrument for control of Hg (UNEP Chemicals Branch 2008). This metal can be found in most ecosystems in three different oxidation forms: metallic (Hg 0 ), monovalent (Hg 2+ 2 ) or divalent (Hg 2+ ), being the last form the most abundant in wellaerated environments. It is found frequently in minerals associated with Cl, OH and reduced sulfur (i.e. cinnabar). Mercury can also be associated with carbon to form chemical species of organic Hg, such as methylhg (CH 3 Hg) or dimethylhg (CH 3 HgCH 3 ), the most toxic and abundant forms. These types of Hg are mainly produced by sulfate-reducing bacteria, and can bioaccumulate and biomagnify in the food chain, principally in the consumption of fish, shellfish, seaweed and marine mammals (Clarkson 1997). Fig. 2A shows the major ecosystem compartments where Hg accumulate, with the inter-conversion of Hg species among them. Mercury can be released to the air, water and soil from both natural and anthropogenic sources (UNEP Chemicals Branch 2008). Natural sources of Hg emissions come from natural weathering of Hg-containing rocks, geothermal and volcanic activities, these last can be separated in cataclysmic volcanoes that have the potential to release large amounts of Hg and alter its concentration in the atmosphere for years, fumes of moderate but more constant eruptions that can affect to the local environment (Nriagu and Becker 2003). On the other hand, the anthropogenic sources are divided in primary and secondary sources. 4 Tesis doctoral Juan Sobrino Plata Fig. 2 A. Mercury cycle in the major compartments of an ecosystem cycle: 1. Emissions of Hg from rock weathering, soils, surface waters, volcanoes and human activities. 2. Circulation through the atmosphere. 3. Deposition of Hg. 4. Conversion into other inorganic and organic forms. 5. Precipitation or bioconversion into more volatile or soluble forms. 6. Re-mobilization into the atmosphere or bioaccumulation in food chains (source: B. Main mechanisms of Hg phytoremediation. Mercury (red circles) can be stabilized (phytostabilization) in the rhizosphere, sequestered and accumulated in harvestable parts of tissue (phytoextraction), or modified into a volatilize form (phytovolatilization; modified from Pilon-Smits 2005). Combustion of coal accounts for a large amount of Hg released in the atmosphere; for instance cement production is also an important source of Hg emissions due to the burning of coal or fuel to heat the materials. An important primary source of Hg is mining, with the processing of different ores that release this metal to the environment. In this respect, we can find the largest Hg-contaminated area of the World in the mining area of Almadén (Spain), as a consequence of cinnabar extraction and processing for centuries (since the days of the Roman Empire until 2003 when the mines were closed). It is estimated that one third of Hg used in the Wo
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