title: Early transcriptional responses to mercury: a role for ethylene in mercury-induced stress.
abstract: Understanding the cellular mechanisms of plant tolerance to mercury (Hg) is important for developing phytoremediation strategies of Hg-contaminated soils. The early responses of alfalfa (Medicago sativa) seedlings to Hg were studied using transcriptomics analysis. A Medicago truncatula microarray was hybridized with high-quality root RNA from M. sativa treated with 3 μM Hg for 3, 6 and 24 h. The transcriptional pattern data were complementary to the measurements of root growth inhibition, lipid peroxidation, hydrogen peroxide (H2 O2 ) accumulation and NADPH-oxidase activity as stress indexes. Of 559 differentially expressed genes (DEGs), 91% were up-regulated. The majority of DEGs were shared between the 3 and 6 h (60%) time points, including the 'stress', 'secondary metabolism' and 'hormone metabolism' functional categories. Genes from ethylene metabolism and signalling were highly represented, suggesting that this phytohormone may be relevant for metal perception and homeostasis. Ethylene-insensitive alfalfa seedlings preincubated with the ethylene signalling inhibitor 1-methylcyclopronene and Arabidopsis thaliana ein2-5 mutants confirmed that ethylene participates in the early perception of Hg stress. It modulates root growth inhibition, NADPH-oxidase activity and Hg-induced apoplastic H2 O2 accumulation. Therefore, ethylene signalling attenuation could be useful in future phytotechnological applications to ameliorate stress symptoms in Hg-polluted plants.
authors.affiliation: Laboratorio de Fisiología Vegetal, Departamento de Biología, Universidad Autónoma de Madrid, 28049, Madrid, Spain.|Laboratorio de Fisiología Vegetal, Departamento de Ciencias Ambientales, Universidad de Castilla-La Mancha, Campus Fábrica de Armas, 45070, Toledo, Spain.
authors.forename: M Belén
authors.initials: MB
authors.lastname: Montero-Palmero
authors.affiliation: Laboratorio de Fisiología Vegetal, Departamento de Biología, Universidad Autónoma de Madrid, 28049, Madrid, Spain.
authors.forename: Amanda
authors.initials: A
authors.lastname: Martín-Barranco
authors.affiliation: Laboratorio de Fisiología Vegetal, Departamento de Ciencias Ambientales, Universidad de Castilla-La Mancha, Campus Fábrica de Armas, 45070, Toledo, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: Escobar
authors.affiliation: Laboratorio de Fisiología Vegetal, Departamento de Biología, Universidad Autónoma de Madrid, 28049, Madrid, Spain.
authors.forename: Luis E
authors.initials: LE
authors.lastname: Hernández
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics*; D005030:Ethylenes / Q000378:metabolism*; D015870:Gene Expression; D017343:Genes, Plant; D006861:Hydrogen Peroxide / Q000378:metabolism; D015227:Lipid Peroxidation; D029902:Medicago / Q000235:genetics* / Q000378:metabolism; D000455:Medicago sativa / Q000235:genetics / Q000378:metabolism; D046913:Medicago truncatula / Q000235:genetics / Q000378:metabolism; D008628:Mercury / Q000378:metabolism / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism; D018384:Oxidative Stress; D010937:Plant Growth Regulators / Q000378:metabolism; D018517:Plant Roots / Q000254:growth & development / Q000378:metabolism; D018749:RNA, Plant; D036226:Seedlings; D015398:Signal Transduction; D013312:Stress, Physiological / Q000235:genetics*; D059467:Transcriptome; D015854:Up-Regulation
chemical_list: D005030:Ethylenes; D010937:Plant Growth Regulators; D018749:RNA, Plant; C036216:ethylene; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D008628:Mercury
keywords: 1-methylcyclopropene (1-MCP); Arabidopsis thaliana; Medicago sativa (alfalfa); ethylene; ethylene insensitive2-5; mercury (Hg); oxidative stress; transcriptome
references.citation: Abercrombie J, Halfhill M, Ranjan P, Rao M, Saxton A, Yuan J, Stewart C. 2008. Transcriptional responses of Arabidopsis thaliana plants to As (V) stress. BMC Plant Biology 8: 87.
references.citation: Agrawal GK, Tamogami S, Han O, Iwahashi H, Rakwal R. 2004. Rice octadecanoid pathway. Biochemical and Biophysical Research Communications 317: 1-15.
references.citation: Agudelo-Romero P, Carbonell P, De La Iglesia F, Carrera J, Rodrigo G, Jaramillo A, Perez-Amador ME, Elena SF. 2008. Changes in the gene expression profile of Arabidopsis thaliana after infection with tobacco etch virus. Virology Journal 5: 92.
references.citation: Arteca RN, Arteca JM. 2007. Heavy-metal-induced ethylene production in Arabidopsis thaliana. Journal of Plant Physiology 164: 1480-1488.
references.citation: Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society. Series B (Methodological) 57: 289-300.
references.citation: Bovet L, Feller U, Martinoia E. 2005. Possible involvement of plant ABC transporters in cadmium detoxification: a cDNA sub-microarray approach. Environment International 31: 263-267.
references.citation: Cao S, Chen Z, Liu G, Jiang L, Yuan H, Ren G, Bian X, Jian H, Ma X. 2009. The Arabidopsis ethylene-insensitive 2 gene is required for lead resistance. Plant Physiology & Biochemistry 47: 308-312.
references.citation: Cargnelutti D, Tabaldi L, Spanevello R, De Oliveira Jucoski G, Battisti V, Redin M, Linares C, Dressler V, De Moraes Flores É, Nicoloso F et al. 2006. Mercury toxicity induces oxidative stress in growing cucumber seedlings. Chemosphere 65: 999-1006.
references.citation: Carrasco-Gil S, Álvarez-Fernández A, Sobrino-Plata J, Millán R, Carpena-Ruiz R, Leduc DL, Andrews JC, Abadía J, Hernández LE. 2011. Complexation of Hg with phytochelatins is important for plant Hg tolerance. Plant, Cell & Environment 34: 778-791.
references.citation: Carrasco-Gil S, Siebner H, LeDuc D, Webb SM, Millan R, Andrews JC, Hernandez LE. 2013. Mercury localization and speciation in plants grown hydroponically or in a natural environment. Environmental Science & Technology 47: 3082-3090.
references.citation: Chen J, Yang ZM. 2012. Mercury toxicity, molecular response and tolerance in higher plants. BioMetals 25: 847-857.
references.citation: Chen L, Song Y, Li S, Zhang L, Zou C, Yu D. 2012a. The role of WRKY transcription factors in plant abiotic stresses. Biochimica et Biophysica Acta 1819: 120-128.
references.citation: Chen CZ, Lv XF, Li JY, Yi HY, Gong JM. 2012b. Arabidopsis NRT1.5 is another essential component in the regulation of nitrate reallocation and stress tolerance. Plant Physiology 159: 1582-1590.
references.citation: Cheng M-C, Liao P-M, Kuo W-W, Lin T-P. 2013. The Arabidopsis ETHYLENE RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals. Plant Physiology 162: 1566-1582.
references.citation: Cho U, Park J. 2000. Mercury-induced oxidative stress in tomato seedlings. Plant Science 156: 1-9.
references.citation: Clèries R, Galvez J, Espino M, Ribes J, Nunes V, de Heredia ML. 2012. BootstRatio: a web-based statistical analysis of fold-change in qPCR and RT-qPCR data using resampling methods. Computers in Biology and Medicine 42: 438-445.
references.citation: Criscuolo G, Valkov VT, Parlati A, Alves LM, Chiurazzi M. 2012. Molecular characterization of the Lotus japonicus NRT1(PTR) and NRT2 families. Plant, Cell & Environment 35: 1567-1581.
references.citation: Cruz-Ortega R, Ownby J. 1993. A protein similar to PR (pathogenesis-related) proteins is elicited by metal toxicity in wheat roots. Physiologia Plantarum 89: 211-219.
references.citation: DalCorso G, Farinati S, Furini A. 2010. Regulatory networks of cadmium stress in plants. Plant Signaling & Behavior 5: 663-667.
references.citation: Desikan R, Hancock JT, Bright J, Harrison J, Weir I, Hooley R, Neill SJ. 2005. A role for ETR1 in hydrogen peroxide signaling in stomatal guard cells. Plant Physiology 137: 831-834.
references.citation: Djebali W, Gallusci P, Polge C, Boulila L, Galtier N, Raymond P, Chaibi W, Brouquisse R. 2008. Modifications in endopeptidase and 20S proteasome expression and activities in cadmium treated tomato (Solanum lycopersicum L.) plants. Planta 227: 625-639.
references.citation: Dong J, Chen C, Chen Z. 2003. Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Molecular Biology 51: 21-37.
references.citation: Douglas-Grubb C, Zipp B, Ludwig-Müller J, Masuno M, Molinski T, Abel S. 2004. Arabidopsis glucosyltransferase UGT74b1 functions in glucosinolate biosynthesis and auxin homeostasis. Plant Journal 40: 893-908.
references.citation: Ernst W, Krauss GJ, Verkleij JAC, Wesengerg D. 2008. Interaction of heavy metals with the sulphur metabolism in angiosperms from an ecological point of view. Plant, Cell & Environment 31: 123-143.
references.citation: Foyer C, Noctor G. 2003. Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiologia Plantarum 119: 355-364.
references.citation: Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K. 2006. Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Current Opinion in Plant Biology 9: 436-442.
references.citation: Glazebrook J. 2005. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annual Review of Phytopathology 43: 205-227.
references.citation: Heaton ACP, Rugh CL, Wang NJ, Meagher RB. 2005. Physiological responses of transgenic merA-tobacco (Nicotiana tabacum) to foliar and root mercury exposure. Water, Air & Soil Pollution 161: 137-155.
references.citation: Heidenreich B, Mayer K, Sandermann HJR, Ernst D. 2001. Mercury-induced genes in Arabidopsis thaliana: identification of induced genes upon long-term mercuric ion exposure. Plant, Cell & Environment 24: 1227-1234.
references.citation: Herbette S, Taconnat L, Hugouvieux V. 2006. Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots. Biochimie 88: 1751-1765.
references.citation: Hernández LE, Ortega-Villasante C, Montero-Palmero MB, Escobar C, Carpena RO. 2012. Heavy metal perception in a microscale environment: a model system using high doses of pollutants. In: Gupta DK, Sandalio LM, eds. Metal toxicity in plants: perception, signalling and remediation. Heidelberg, Germany: Springer, 23-39.
references.citation: Heyno E, Klose C, Krieger-Liszkay A. 2008. Origin of cadmium-induced reactive oxygen species production: mitochondrial electron transfer versus plasma membrane NADPH-oxidase. New Phytologist 179: 687-699.
references.citation: Javot H, Maurel C. 2002. The role of aquaporins in root water uptake. Annals of Botany 90: 301-313.
references.citation: Küster H, Becker A, Firnhaber C, Hohnjec N, Manthey K, Perlick AM, Bekel T, Dondrup M, Henckel K, Goesmann A et al. 2007. Development of bioinformatic tools to support EST-sequencing, in silico- and microarray-based transcriptome profiling in mycorrhizal symbioses. Phytochemistry 68: 19-32.
references.citation: Laemmli L. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
references.citation: Lei G, Shen M, Li ZG, Zhang B, Duan KX, Wang N, Cao YR, Zhang WK, Ma B, Ling HQ et al. 2011. EIN2 regulates salt stress response and interacts with a MA3 domain containing protein ECIP1 in Arabidopsis. Plant, Cell & Environment 34: 1678-1692.
references.citation: Lequeux H, Hermans C, Lutts S, Verbruggen N. 2010. Response to copper excess in Arabidopsis thaliana: impact on the root system architecture, hormone distribution, lignin accumulation and mineral profile. Plant Physiology & Biochemistry 48: 673-682.
references.citation: Li P, Feng XB, Qiu GL, Shang LH, Li ZG. 2009. Mercury pollution in Asia: a review of the contaminated sites. Journal of Hazardous Materials 168: 591-601.
references.citation: Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using Real-Time Quantitative PCR and the 2−ΔΔCT. Methods 25: 402-408.
references.citation: Maksymiec W, Krupa Z. 2006. The effects of short-term exposition to Cd, excess Cu ions and jasmonate on oxidative stress appearing in Arabidopsis thaliana. Environmental & Experimental Botany 57: 187-194.
references.citation: Maksymiec W, Wianowska D, Dawidowicz AL, Radkiewicz S, Mardarowicz M, Krupta Z. 2005. The level of jasmonic acid in Arabidopsis thaliana and Phaseolus coccineus plants under heavy metal stress. Journal of Plant Physiology 162: 1338-1346.
references.citation: Mersmann S, Bourdais G, Rietz S, Robatzek S. 2010. Ethylene signaling regulates accumulation of the FLS2 receptor and is required for the oxidative burst contributing to plant immunity. Plant Physiology 154: 391-400.
references.citation: Mittler R. 2006. Abiotic stress, the field environment and stress combination. Trends in Plant Science 11: 15-19.
references.citation: Moons A. 2003. Ospdr9, which encodes a PDR-type ABC transporter, is induced by heavy metals, hypoxic stress and redox perturbations in rice roots. FEBS Letters 553: 370-376.
references.citation: Nocito F, Espen L, Crema B, Cocucci M, Sacchi G. 2008. Cadmium induces acidosis in maize root cells. New Phytologist 179: 700-711.
references.citation: Norton G-J, Lou-Hing D-E, Meharg A, Price A. 2008. Rice-arsenate interactions in hydroponics: whole genome transcriptional analysis. Journal of Experimental Botany 59: 2267-2276.
references.citation: Ortega-Villasante C, Hernández LE, Rellán-Álvarez R, Del Campo FF, Carpena-Ruiz RO. 2007. Rapid alteration of cellular redox homeostasis upon exposure to cadmium and mercury in alfalfa seedlings. New Phytologist 176: 96-107.
references.citation: Ortega-Villasante C, Rellán-Álvarez R, Del Campo FF, Carpena-Ruiz RO, Hernández LE. 2005. Cellular damage induced by cadmium and mercury in Medicago sativa. Journal of Experimental Botany 56: 2239-2251.
references.citation: Patra M, Bhowmik N, Bandopadhyay B, Sharma A. 2004. Comparison of mercury, lead and arsenic with respect to genotoxic effects of plant systems and the development of genetic tolerance. Environmental & Experimental Botany 52: 199-223.
references.citation: Patra M, Sharma A. 2000. Mercury toxicity in plants. The Botanical Review 66: 379-422.
references.citation: Pitta-Alvarez S, Spollansky T, Giulietti A. 2000. The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida. Enzyme & Microbial Technology 26: 252-258.
references.citation: Portillo M, Fenoll C, Escobar C. 2006. Evaluation of different RNA extraction methods for small quantities of plant tissue: combined effects of reagent type and homogenization procedure on RNA quality-integrity and yield. Physiologia Platarum 128: 1-7.
references.citation: Rellán-Álvarez R, Ortega-Villasante C, Álverez-Fernández A, del Campo FF, Hernández LE. 2006. Stress responses of Zea mays to cadmium and mercury. Plant and Soil 279: 41-50.
references.citation: Rodríguez-Serrano M, Romero-Puertas M, Pazmiño D, Testillano P, Risueño M, Luis A, Sandalio L. 2009. Cellular response of pea plants to cadmium toxicity: cross talk between reactive oxygen species, nitric oxide, and calcium. Plant Physiology 150: 229-243.
references.citation: Romero-Puertas M, Rodríguez-Serrano M, Corpas F, Gomez M, Del Rio L, Sandalio L. 2004. Cadmium induced subcellular accumulation of O2 and H2O2 in pea leaves. Plant, Cell & Environment 27: 1122-1134.
references.citation: Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, Li J, Thiagarajan M, White JA, Quackenbush J. 2006. TM4 microarray software suite. Methods in Enzymology 411: 134-193.
references.citation: Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M et al. 2003. TM4: a free, open-source system for microarray data management and analysis. BioTechniques 34: 374-378.
references.citation: Sagi M, Fluhr R. 2001. Superoxide production by plant homologues of the gp91phox NADPH-oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection. Plant Physiology 126: 1281-1290.
references.citation: Sävenstrand H, Strid Å. 2004. Six genes strongly regulated by mercury in Pisum sativum roots. Plant Physiology & Biochemistry 42: 135-142.
references.citation: Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T. 2006. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology 7: 3.
references.citation: Schneider CA, Rasband WS, Eliceiri KW. 2012. National Institutes of Health Image to ImageJ: 25 years of image analysis. Nature Methods 9: 671-675.
references.citation: Seth CS, Remans T, Keunen E, Jozefczak M, Gielen H, Opdenakker K, Weyens N, Vangronsveld J, Cuypers A. 2012. Phytoextraction of toxic metals: a central role for glutathione. Plant, Cell & Environment 35: 334-346.
references.citation: Sobrino-Plata J, Ortega-Villasante C, Flores-Cáceres ML, Escobar C, Del Campo FF, Hernandez LE. 2009. Differential alterations of antioxidant defenses as bioindicators of mercury and cadmium toxicity in alfalfa. Chemosphere 77: 946-954.
references.citation: Sudo E, Itouga M, Yoshida-Hatanaka K, Ono Y, Sakakibara H. 2008. Gene expression and sensitivity in response to copper stress in rice leaves. Journal of Experimental Botany 59: 3465-3474.
references.citation: Sun P, Tian Q, Chen J, Zhang W. 2010. Aluminium-induced inhibition of root elongation in Arabidopsis is mediated by ethylene and auxin. Journal of Experimental Botany 61: 347-356.
references.citation: Swarup R, Perry P, Hagenbeek D, Van Der Straeten D, Beemster GTS, Sandberg G, Bhalerao R, Ljung K, Bennett MJ. 2007. Ethylene up regulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation. The Plant Cell 19: 2186-2196.
references.citation: Swindell WR, Huebner M, Weber A. 2007. Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genomics 8: 125.
references.citation: Thapa G, Sadhukhan A, Panda SK, Sahoo L. 2012. Molecular mechanistic model of plant heavy metal tolerance. BioMetals 25: 489-505.
references.citation: Thimm O, Blasing O, Gibon Y, Nagel A, Meyer S, Kruger P, Selbig J, Muller LA, Rhee SY, Stitt M. 2004. MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant Journal 37: 914-939.
references.citation: Thomma BPHJ, Eggermont K, Tierens KFMJ, Broekaert WF. 1999. Requirement of functional Ethylene-Insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea. Plant Physiology 121: 1093-1101.
references.citation: Tocquin P, Corbesier L, Havelange A, Pieltain A, Kurtem E, Bernier G, Perilleux C. 2003. A novel high efficiency, low maintenance, hydroponic system for synchronous growth and flowering of Arabidopsis thaliana. BMC Plant Biology 3: 2.
references.citation: Torres MA, Dangl JL, Jones JDG. 2002. Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proceedings of the National Academy of Sciences, USA 99: 517-522.
references.citation: Torres MA, Jones JDG, Dangl JL. 2005. Pathogen-induced, NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana. Nature Genetics 37: 1130-1134.
references.citation: Van De Mortel J, Henk S, Moerland P, Themaat E, Van Der Ent S, Blankestijn H, Ghandilyan A, Tsiatsiani S, Aarts M. 2008. Expression differences for genes involved in lignin, glutathione and sulphate metabolism in response to cadmium in Arabidopsis thaliana and the related Zn/Cd hyperaccumulator Thlaspi caerulescens. Plant, Cell & Environment 31: 301-324.
references.citation: Wang Y, Liu C, Li K, Sun F, Hu H, Li X, Zhao Y, Han C, Zhang W, Duan Y. 2007. Arabidopsis EIN2 modulates stress response through abscisic acid response pathway. Plant Molecular Biology 64: 633-644.
references.citation: Wang KLC, Li H, Ecker JR. 2002. Ethylene biosynthesis and signaling networks. The Plant Cell 14(Suppl): S131-S151.
references.citation: Weber M, Trampczynska A, Clemens S. 2006. Comparative transcriptome analysis of toxic metal responses in Arabidopsis thaliana and the Cd2+-hypertolerant facultative metallophyte Arabidopsis halleri. Plant, Cell & Environment 29: 950-963.
references.citation: Xiang C, Oliver DJ. 1998. Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. The Plant Cell 10: 1539-1550.
references.citation: Xu J, Su J, Du L, Liu X. 2012. Comparative transcriptome analysis of cadmium responses in Solanum nigrum and Solanum torvum. New Phytologist 196: 110-124.
references.citation: Yastreb TO, Kolupaev YY, Vayner AO. 2012. Induction of heat resistance in wheat coleoptiles by 4-hydroxybenzoic acid: connection with the generation of reactive oxygen species. Journal of Stress Physiology & Biochemistry 8: 72-81.
references.citation: Zhao FY, Han MM, Zhang SY, Wang K, Zhang CR, Liu T, Liu W. 2012. Hydrogen peroxide-mediated growth of the root system occurs via auxin signaling modification and variations in the expression of cell-cycle genes in rice seedlings exposed to cadmium stress. Journal of Integrative Plant Biology 54: 991-1006.
references.citation: Zhou ZS, Huang SQ, Guo K, Mehta SK, Zhang PC, Yang ZM. 2007. Metabolic adaptations to mercury-induced oxidative stress in roots of Medicago sativa L. Journal of Inorganic Biochemistry 101: 1-9.
references.citation: Zhou ZS, Wang SJ, Yang ZM. 2008. Biological detection and analysis of mercury toxicity to alfalfa (Medicago sativa) plants. Chemosphere 70: 1500-1509.
references.citation: Zhou ZS, Yang SN, Li H, Zhu CC, Liu ZP, Yang ZM. 2013. Molecular dissection of mercury-responsive transcriptome and sense/antisense genes in Medicago truncatula by high-throughput sequencing. Journal of Hazardous Materials 252: 123-131.