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Early physiological and biochemical responses of rice seedlings to low concentration of microcystin-LR

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Abstract

Microcystin-leucine and arginine (microcystin-LR) is a cyanotoxin produced by cyanobacteria like Microcystis aeruginosa, and it’s considered a threat to water quality, agriculture, and human health. Rice (Oryza sativa) is a plant of great importance in human food consumption and economy, with extensive use around the world. It is therefore important to assess the possible effects of using water contaminated with microcystin-LR to irrigate rice crops, in order to ensure a safe, high quality product to consumers. In this study, 12 and 20-day-old plants were exposed during 2 or 7 days to a M. aeruginosa extract containing environmentally relevant microcystin-LR concentrations, 0.26–78 μg/L. Fresh and dry weight of roots and leaves, chlorophyll fluorescence, glutathione S-transferase and glutathione peroxidase activities, and protein identification by mass spectrometry through two-dimensional gel electrophoresis from root and leaf tissues, were evaluated in order to gauge the plant’s physiological condition and biochemical response after toxin exposure. Results obtained from plant biomass, chlorophyll fluorescence, and enzyme activity assays showed no significant differences between control and treatment groups. However, proteomics data indicates that plants respond to M. aeruginosa extract containing environmentally relevant microcystin-LR concentrations by changing their metabolism, responding differently to different toxin concentrations. Biological processes most affected were related to protein folding and stress response, protein biosynthesis, cell signalling and gene expression regulation, and energy and carbohydrate metabolism which may denote a toxic effect induced by M. aeruginosa extract and microcystin-LR. The implications of the metabolic alterations in plant physiology and growth require further elucidation.

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References

  • Asensi M, Sastre J, Pallardo FV, Lloret A, Lehner M, Garcia de la Assuncion J (1999) Ration of reduced to oxidized glutathione as indicator of oxidative stress status and DNA damage. Method Enzymol 299:267–276. doi:10.1016/S0076-6879(99)99026-2

    Article  CAS  Google Scholar 

  • Baker NR, Rosenqvist E (2004) Applications of chlorophyll fluorescence can improve crop production strategies: an examination of future possibilities. J Exp Bot 55:1607–1621. doi:10.1093/jxb/erh196

    Article  CAS  Google Scholar 

  • Bibo L, Yan G, Bangding X, Jiantong L, Yongding L (2008) A laboratory study on risk assessment of microcystin-RR in cropland. J Environ Manage 86:566–574. doi:10.1016/j.jenvman.2006.12.040

    Article  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3

    Article  CAS  Google Scholar 

  • Campos A, da Costa G, Coelho AV, Fevereiro P (2009) Identification of bacterial protein markers and enolase as a plant response protein in the infection of Olea europaea subsp.europaea by Pseudomonas savastanoi pv. savastanoi. Eur J Plant Pathol 125:603–616. doi:10.1007/s10658-009-9509-0

    Article  CAS  Google Scholar 

  • Campos A, Puerto M, Prieto A, Cameán A, Almeida AM, Coelho AV, Vasconcelos V (2012) Protein extraction and two-dimensional gel electrophoresis of proteins in the marine mussel Mytilus galloprovincialis: an important tool for protein expression studies, food quality and safety assessment. J Sci Food Agric 93:1779–1787. doi:10.1002/jsfa.5977

    Article  Google Scholar 

  • Campos A, Araújo P, Pinheiro C, Azevedo J, Osório H, Vasconcelos V (2013) Effects on growth, antioxidant enzyme activity and levels of extracellular proteins in the green alga Chlorella vulgaris exposed to crude cyanobacterial extracts and pure microcystin and cylindrospermopsin. Ecotox Environ Saf 94:45–53. doi:10.1016/j.ecoenv.2013.04.019

    Article  CAS  Google Scholar 

  • Chen J, Song L, Dai J, Gan N, Zhili L (2004) Effects of microcystins on the growth and the activity of superoxide dismutase and peroxidade of rape (Brassica napus L.) and rice (Oryza sativa L.). Toxicon 43:393–400. doi:10.1016/j.toxicon.2004.01.011

    Article  CAS  Google Scholar 

  • Chen J, Han FX, Wang F, Haiqiang Z, Shi Z (2012) Accumulation and phytotoxicity of microcystin-LR in rice (Oryza sativa). Ecotox Environ Saf 76:193–199. doi:10.1016/j.ecoenv.2011.09.022

    Article  CAS  Google Scholar 

  • Crush JR, Briggs LR, Sprosen JM, Nichols SN (2008) Effect of irrigation with lake water containing microcystins on microcystin content and growth of ryegrass, clover, rape, and lettuce. Environ Toxicol 23:246–252. doi:10.1002/tox.20331

    Article  CAS  Google Scholar 

  • Dawson RM (1998) The toxicology of microcystins. Toxicon 36:953–962. doi:10.1016/S0041-0101(97)00102-5

    Article  CAS  Google Scholar 

  • El Khalloufi F, Oufdou K, Lahrouni M, El Ghazali I, Saqrane S, Vasconcelos V, Oudra B (2011) Allelopatic effects of cyanobacteria extracts containing microcystins on Medicago sativa-Rhizobia symbiosis. Ecotox Environ Saf 74(431):438. doi:10.1016/j.ecoenv

    Google Scholar 

  • El Khalloufi F, El Ghazali I, Saqrana S, Oufdou K, Vasconcelos V, Oudra B (2012) Phytotoxic effects of a natural bloom extract containing microcystons on Lycopersicon esculentum. Ecotox Environ Saf 79:199–205. doi:10.1016/j.ecoenv.2012.01.002

    Article  Google Scholar 

  • Eshdat Y, Holland D, Faltin Z, Ben-Hayyim G (1997) Plant glutathione peroxidases. Physiol Plant 100:234–240. doi:10.1111/j.1399-3054.1997.tb04779.x

    Article  CAS  Google Scholar 

  • Faltin Z, Holland D, Velcheva M, Tsapovestky M, Roeckel-Drevet P, Handa AK, Mohamad A–A, Friedman-Einat M, Eshdat Y, Perl A (2010) Glutathione peroxidase regulation of reactive oxygen species level is crucial for in vitro plant differentiation. Plant Cell Physiol 51:1151–1162. doi:10.1093/pcp/pcq082

    Article  CAS  Google Scholar 

  • Flohé L, Gunzler WA (1984) Assays of glutathione peroxidase. Method Enzimol 105:114–121

    Article  Google Scholar 

  • Gobom J, Nordhoff E, Mirgorodskaya E, Ekman R, Roepstorff P (1999) Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom 34:105–116. doi:10.1002/(SICI)1096-9888(199902)34:2<105:AID-JMS768>3.0.CO;2-4

    Article  CAS  Google Scholar 

  • Gomes C, Almeida A, Ferreira JA, Silva L, Santos-Sousa H, Pinto-de-Sousa J, Santos LL, Amado F, Schwientek T, Levery SB, Mandel U, Clausen H, David L, Reis CA, Osório H (2013) Glycoproteomic analysis of serum from patients with gastric precancerous lesions. J Proteome Res 12:1454–1466. doi:10.1021/pr301112x

    Article  CAS  Google Scholar 

  • Habig W, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases, the first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139

    CAS  Google Scholar 

  • Heinemeyer J, Lewejohann D, Braun HP (2007) Blue-native gel electrophoresis for the characterization of protein complexes in plants. Methods Mol Biol 335:343–352. doi:10.1385/1-59745-227-0:343

    Google Scholar 

  • Hitzfeld BC, Hoger SJ, Dietrich DR (2000) Cyanobacterial toxins: removal during drinking water treatment, and human risk assessment. Environ Health Perspect 108:113–122

    CAS  Google Scholar 

  • Ibelings BW, Chorus I (2007) Accumulation of cyanobacterial toxins in freshwater ‘‘seafood’’ and its consequences for public health: a review. Environ Pollut 150:177–192. doi:10.1016/j.envpol.2007.04.012

    Article  CAS  Google Scholar 

  • Jámbrik K, Máthé C, Vasas G, Beyer D, Molnár E, Borbély G, M-Hamvas M (2011) Microcystin-LR induces chromatin alterations and modulates neutral single-strand-preferring nuclease activity in Phragmites australis. J Plant Physiol 168:678–686. doi:10.1016/j.jplph.2010.10.007

    Article  Google Scholar 

  • Kótai J (1972) Instructions for preparation of modified nutrient solution Z8 for algae. Norwegian Institute for Water Research, Oslo

    Google Scholar 

  • Lahrouni M, Oufdou K, Faghire M, Peix A, El Khalloufi F, Vasconcelos V, Oudra B (2012) Cyanobacterial extracts containing microcystins affect the growth, nodulation process and nitrogen uptake of faba bean (Vicia faba L., Fabaceae). Ecotoxicology 21:681–687. doi:10.1007/s10646-011-0826-7

    Article  CAS  Google Scholar 

  • Lambert TW, Boland MP, Holmes CFB, Hrudey SE (1994) Quantitation of the Microcystin hepatotoxins in water at environmentally relevant concentrations with the Protein Phosphatase Bioassay. Environ Sci Technol 28:753–755. doi:10.1021/es00053a032

    Article  CAS  Google Scholar 

  • Lawrence RA, Burk RF (1976) Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun 71:952–958. doi:10.1016/0006-291X(76)90747-6

    Article  CAS  Google Scholar 

  • Lee R, Britz-McKibbin P (2009) Differential rates of glutathione oxidation for assessment of cellular redox status and antioxidant capacity by capillary electrophoresis-mass spectrometry: an elusive biomarker of oxidative stress. Anal Chem 81:7047–7056. doi:10.1021/ac901174g

    Article  CAS  Google Scholar 

  • Lee J–J, Jo H-J, Kong K-H (2011) A plant-specific Tau class glutathione S-transferase from Oryza sativa having significant detoxification activity towards chloroacetanilide herbicides. Bull Korean Chem Soc 32:3756–3759. doi:10.1016/j.pestbp.2011.10.005

    Article  CAS  Google Scholar 

  • Levison BS, Zhang R, Wang Z, Fu X, DiDonato JA, Hazen SL (2013) Quantification of fatty acid oxidation products using online high-performance liquid chromatography tandem mass spectrometry. Free Radic Biol Med 59:2–13. doi:10.1016/j.freeradbiomed.2013.03.001

    Article  CAS  Google Scholar 

  • Marrs KA (1996) The functions and regulation of glutathione S-transferases in plants. Annu Rev Plant Physiol Plant Mol Biol 47:127–158. doi:10.1146/annurev.arplant.47.1.127

    Article  CAS  Google Scholar 

  • Máthé C, Beyer D, Erdodi F, Serfozo Z, Székyölgyi L, Vasas G, M-Hamyas M, Jámbrik K, Gonda S, Kiss A, Szigeti ZM, Surányi G (2009) Microcystin-LR induces abnormal root development by altering microtubuleorganization in tissue-cultured common reed (Phragmites australis) plantlets. Aquat Toxicol 92:122–130. doi:10.1016/j.aquatox.2009.02.005

    Article  Google Scholar 

  • Maxwell K, Johnson N (2000) Chlorophyll fluorescence – a practical guide. J Exp Bot 51:659–668. doi:10.1093/jexbot/51.345.659

    Article  CAS  Google Scholar 

  • Miao Y, Lv D, Wang P, Wang X-C, Chen J, Miao C, Song C-P (2006) An arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress responses. Plant Cell 18:2749–2766. doi:10.1105/tpc.106.044230

    Article  CAS  Google Scholar 

  • Miché L, Balandreau J (2001) Effects of rice seed surface sterilization with hypochlorite on inoculated Burkholderia vietnamiensis. Appl Environ Microbiol 67:3046–3052. doi:10.1128/AEM.67.7.3046- 3052.2001

    Article  Google Scholar 

  • Navrot N, Collin V, Gualberto J, Gelhaye E, Hirasawa M, Rey P, Knaff DB, Isaakidis E, Jacquot J-P, Rouhier N (2006) Plant glutathione peroxidases are functional peroxiredoxins distributed in several subcellular compartments and regulated during biotic and abiotic stresses. Plant Physiol 142:1364–1379. doi:10.1104/pp.106.089458

    Article  CAS  Google Scholar 

  • Neuhoff V, Arnold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255–262. doi:10.1002/elps.1150090603

    Article  CAS  Google Scholar 

  • Oh H-M, Lee SJ, Kim J-H, Kim H-S, Yoon B-D (2001) Seasonal variation and indirect monitoring of microcystin concentrations in Daechung reservoir, Korea. Appl Environ Microbiol 67:1484–1489. doi:10.1128/AEM.67.4.1484-1489.2001

    Article  CAS  Google Scholar 

  • Perkins DN, Pappin Dj, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567. doi:10.1002/(SICI)1522-2683(19991201)20

    Article  CAS  Google Scholar 

  • Peuthert A, Chakrabarti S, Pflugmacher S (2007) Uptake of microcystins-LR and –LF (cyanobacterial toxins) in seedlings of several important agricultural plant species and the correlation with cellular damage (lipid peroxidation). Environ Toxicol 22(436):442. doi:10.1002/tox.20266

    Google Scholar 

  • Peuthert A, Lawton L, Pflugmacher S (2008) In vivo influence of cyanobacterial toxins on enzyme activity and gene expression of protein phosphatases in Alfalfa (Medicago sativa). Toxicon 52:84–90. doi:10.1016/j.toxicon.2008.04.172

    Article  CAS  Google Scholar 

  • Pflugmacher S (2004) Promotion of oxidative stress in the aquatic macrophyte Ceratophyllum demersum during biotransformation of the cyanobacterial toxin Microcystin-LR. Aquat Toxicol 70:169–178. doi:10.1016/j.aquatox.2004.06.010

    Article  CAS  Google Scholar 

  • Pflugmacher S, Aulhorn M, Grimm B (2007) Influence of a cyanobacterial crude extract containing microcystin-LR on the physiology and antioxidative defence systems of different spinach variants. New Phytol 175:482–489. doi:10.1111/j.1469-8137.2007.02144.x

    Article  CAS  Google Scholar 

  • Pinheiro C, Azevedo J, Campos A, Loureiro S, Vasconcelos V (2013) Absence of negative allelopathic effects of cylindrospermopsin and microcystin-LR on selected marine and freshwater phytoplankton species. Hydrobiologia 705:27–42. doi:10.1007/s10750-012-1372-x

    Article  CAS  Google Scholar 

  • Prieto A, Campos A, Cameán A, Vasconcelos V (2011) Effects on growth and oxidative stress status of rice plants (Oryza sativa) exposed to two extracts of toxin-producing cyanobacteria (Aphanizomenon ovalisporum and Microcystis aeruginosa). Ecotoxicology and Enviromental Safety 74:1973–1980. doi:10.1016/j.ecoenv.2011.06.009

    Article  CAS  Google Scholar 

  • Ramanan S, Tang J, Velayudhan A (2000) Isolation and preparative purification of microcystin variants. J Chromatogr A 883:103–112. doi:10.1016/S0021-9673(00)00378-2

    Article  CAS  Google Scholar 

  • Rivasseau C, Racaud P, Deguin A, Hennion M-C (1999) Development of a bioanalytical phosphatase inhibition test for the monitoring of microcystins in environmental water samples. Anal Chim Acta 394:243–257. doi:10.1016/S0003-2670(99)00301-3

    Article  CAS  Google Scholar 

  • Saker ML, Fastner J, Dittmann E, Christiansen G, Vasconcelos VM (2005) Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river. J Appl Microbiol 99:749–757. doi:10.1111/j.1365-2672.2005.02687.x

    Article  CAS  Google Scholar 

  • Saqrane S, El Ghazali I, Ouahid Y, El Hassni M, El Hadrami I, Bouarad L, del Campo FF, Oudra B, Vasconcelos V (2007) Phytotoxic effects of cyanobacteria extract on the aquatic plant Lemna gibba: microcystin accumulation, detoxication and oxidative stress induction. Aquat Toxicol 83:284–294. doi:10.1016/j.aquatox.2007.05.004

    Article  CAS  Google Scholar 

  • Saqrane S, Ghazali IE, Oudra B, Bouarab L, Vasconcelos V (2008) Effects of cyanobacteria producing microcystins on seed germination and seedling growth of several agricultural plants. J Environ Sci Health, Part B 43:443–451. doi:10.1080/10934520701796192

    Article  CAS  Google Scholar 

  • Saqrane S, Ouahid Y, El Ghazali I, Oudra B, Bouarab L, del Campo FF (2009) Physiological changes in Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars, caused by irrigation with water contaminated with microcystins: a laboratory experimental approach. Toxicon 53:786–796. doi:10.1016/j.toxicon.2009.01.028

    Article  CAS  Google Scholar 

  • Sivonen K, Jones G, Chorus I, Bartram J (1999) Toxic cyanobacteria in water: a guide to their public health consequences, monitoring and management. E&FN Spon, London

    Google Scholar 

  • Tyler AN, Hunter PD, Carvalho L, Codd GA, Elliott JA, Ferguson CA, Hanley ND, Hopkins DW, Maberly SC, Mearns KJ, Scott EM (2009) Strategies for monitoring and managing mass populations of toxic cyanobacteria in recreational waters: a multi-interdisciplinary approach. Environ Health 8:S11. doi:10.1186/1476-069X-8-S1-S11

    Article  Google Scholar 

  • van Apeldoorn ME, van Egmond HP, Speijers GJA, Bakker GJI (2007) Toxins of cyanobacteria. Mol Nutr Food Res. doi:10.1002/mnfr.200600185

    Google Scholar 

  • Vinagre C, Madeira D, Narciso L, Cabral HN, Diniz M (2012) Effect of temperature on oxidative stress in fish: lipid peroxidation and catalase activity in the muscle of juvenile seabass, Dicentrarchus labrax. Ecol Indic 23:274–279. doi:10.1016/j.ecolind.2012.04.009

    Article  CAS  Google Scholar 

  • Wang W, Vinocur B, Shoseyov O, Altman A (2004) Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci 9:244–252. doi:10.1016/j.tplants.2004.03.006

    Article  CAS  Google Scholar 

  • WHO (2003) Cyanobacterial toxins: Microcystin-LR in drinking-water. Background document for preparation of WHO Guidelines for drinking-water quality. World Health Organization. http://www.who.int/water_sanitation_health/dwq/en/gdwq3_12.pdf Accessed 14 May 2011

  • Xiao FG, Zhao XL, Tang J, Gu XH, Zhang JP, Niu WM (2009) Necessity of screening water chestnuts for microcystins after cyanobacterial blooms break out. Arch Environ Contam Toxicol 57:256–263. doi:10.1007/s00244-008-9275-6

    Article  CAS  Google Scholar 

  • Yin L, Huang J, Li D, Liu Y (2005) Microcystin-RR uptake and its effects on the growth of submerged macrophyte Vallisneria natans (lour.). Environ Toxicol 20:308–313. doi:10.1002/tox.20122

    Article  CAS  Google Scholar 

  • Yoshida S, Forno DA, Cock JH, Gomez KA (1976) Laboratory manual for physiological studies of rice, 3rd edn. IRRI, Manila, pp 61–66

    Google Scholar 

  • Zhang H-j, Zhang J-y, Hong Y, Chen Y-x (2007) Evaluation of organ distribution of microcystins in the freshwater phytoplanktivorous fish Hypophthalmichthys molitrix. J Zhejiang Univ Sci B 8:116–120. doi:10.1631/jzus.2007.B0116

    Article  Google Scholar 

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Acknowledgments

Alexandre Campos and Hugo Osório’s work contracts are supported by the Ciência 2007 program of the Ministério da Educação e Ciência (MEC, Lisbon, Portugal). This work was supported by the projects PP-IJUP2O11-3 from Porto University and PEst-C/MAR/LA0015/2011 from Foundation for Science and Technology (FCT) and by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme. The authors thank Sociedade Europeia de Arroz (SEAR, SA) for kindly providing the rice seeds in this study.

Ethical standards

The authors declare that the species utilized in this work are not included in the European Union Legislation for the protection of animals used for scientific purposes (Directive 2010/63/EU).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Centro Interdisciplinar de Investigação Marinha e Ambiental, CIIMAR/CIMAR, Rua dos Bragas 289, 4050-123, Porto, Portugal

    Catarina C. Azevedo, Joana Azevedo, Vitor Vasconcelos & Alexandre Campos

  2. ESTSP - School of Health and Technology of Porto, Polytechnic Institute of Porto, 4400-330, Vila Nova de Gaia, Portugal

    Joana Azevedo

  3. Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal

    Hugo Osório

  4. Faculty of Medicine, University of Porto, Porto, Portugal

    Hugo Osório

  5. Department of Biology, Faculty of Sciences of the University of Porto, Porto, Portugal

    Vitor Vasconcelos

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  1. Catarina C. Azevedo
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  2. Joana Azevedo
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  3. Hugo Osório
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  4. Vitor Vasconcelos
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  5. Alexandre Campos
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Correspondence to Alexandre Campos.

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Azevedo, C.C., Azevedo, J., Osório, H. et al. Early physiological and biochemical responses of rice seedlings to low concentration of microcystin-LR. Ecotoxicology 23, 107–121 (2014). https://doi.org/10.1007/s10646-013-1156-8

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