Abstract
Various fluorescence reagents are often used for the detection of oxidative stress, but more extensive comparison in the same study is rather rare. We tested five ROS-related and two non-ROS-related reagents in the roots of Zea mays germinated and growing in 1 or 100 µM cadmium-enriched solution. Namely, “general” ROS (CellROX Deep Red reagent and 2′,7′-dichlorodihydrofluorescein diacetate), hydrogen peroxide (Amplex UltraRed), superoxide radical (dihydroethidium), hydroxyl radical/peroxynitrite (aminophenyl fluorescein), nitric oxide (1,2-diaminoanthraquinone), and glutathione/-SH (monochlorobimane) indicators were tested. Both Cd doses stimulated signal of various ROS often extensively compared to control and mainly H2O2 differed between 1 and 100 µM Cd. Signal of ROS was clearly visible in exodermis and vascular tissue through which cadmium is transported via transpiration stream. CellROX Deep Red reagent gives bright and clear signal on the root cross sections. The standard spectrophotometry (detection of H2O2 and superoxide) did not reveal differences between control and 1 µM Cd, indicating that fluorescence microscopy is more sensitive to detect low change in oxidative balance. Nitric oxide and glutathione/-SH signal was less different between control and Cd treatments, indicating their contribution to metal tolerance. Cd accumulation and translocation and the use of reagents under the excess of other metals are also discussed.
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References
Anjum SA, Tanveer M, Hussain S, Bao M, Wang L, Khan I, Ullah E, Tung SA, Samad RA, Shahzad B (2015) Cadmium toxicity in Maize (Zea mays L.): consequences on antioxidative systems, reactive oxygen species and cadmium accumulation. Environ Sci Pollut Res 22:17022–17030
Babula P, Klejdus B, Kováčik J, Hedbavny J, Hlavna M (2015) Lanthanum rather than cadmium induces oxidative stress and metabolite changes in Hypericum perforatum. J Hazard Mater 286:334–342
Chen Y, Mo H-Z, Zheng M-Y, Xian M, Qi Z-Q, Li Y-Q, Hu L-B, Chen J, Yang L-F (2014) Selenium inhibits root elongation by repressing the generation of endogenous hydrogen sulfide in Brassica rapa. PLoS One 9(10):e110904. doi:10.1371/journal.pone.0110904
Kalyanaraman B, Darley-Usmar V, Davies KJA, Dennery PA, Forman HJ, Grisham MB, Mann GE, Moore K, Jackson Roberts L II, Ischiropoulos H (2012) Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic Biol Med 52:1–6
Kováčik J (2013) Hyperaccumulation of cadmium in Matricaria chamomilla: a never-ending story? Acta Physiol Plant 35:1721–1725
Kováčik J, Bačkor M (2008) Oxidative status of Matricaria chamomilla plants related to cadmium and copper uptake. Ecotoxicology 17:471–479
Kováčik J, Babula P, Hedbavny J, Klejdus B (2014a) Hexavalent chromium damages chamomile plants by alteration of antioxidants and its uptake is prevented by calcium. J Hazard Mater 273:110–117
Kováčik J, Babula P, Hedbavny J, Švec P (2014b) Manganese-induced oxidative stress in two ontogenetic stages of chamomile and amelioration by nitric oxide. Plant Sci 215–216:1–10
Kováčik J, Babula P, Klejdus B, Hedbavny J, Jarošová M (2014c) Unexpected behavior of some nitric oxide modulators under cadmium excess in plant tissue. PLoS One 9(3):e91685. doi:10.1371/journal.pone.0091685
Kováčik J, Štěrbová D, Babula P, Švec P, Hedbavny J (2014d) Toxicity of naturally-contaminated manganese soil to selected crops. J Agric Food Chem 62:7287–7296
Kováčik J, Babula P, Hedbavny J, Kryštofova O, Provaznik I (2015) Physiology and methodology of chromium toxicity using alga Scenedesmus quadricauda as model object. Chemosphere 120:23–30
Kumar P, Tewari RK, Sharma PN (2008) Cadmium enhances generation of hydrogen peroxide and amplifies activities of catalase, peroxidases and superoxide dismutase in maize. J Agron Crop Sci 194:72–80
Liszkay A, van der Zalm E, Schopfer P (2004) Production of reactive oxygen intermediates (O ·−2 , H2O2 and ·OH) by maize roots and their role in wall loosening and elongation growth. Plant Physiol 136:3114–3123
Mihaličová Malčovská S, Dučaiová Z, Maslaňáková I, Bačkor M (2014) Effect of silicon on growth, photosynthesis, oxidative status and phenolic compounds of maize (Zea mays L.) grown in cadmium excess. Water Air Soil Pollut. doi:10.1007/s11270-014-2056-0
Muschitz A, Riou C, Mollet J-C, Gloaguen V, Faugeron C (2015) Modifications of cell wall pectin in tomato cell suspension in response to cadmium and zinc. Acta Physiol Plant. doi:10.1007/s11738-015-2000-y
Poborilova Z, Ohlsson AB, Berglund T, Vildova A, Provaznik I, Babula P (2015) DNA hypomethylation concomitant with the overproduction of ROS induced by naphthoquinone juglone on tobacco BY-2 suspension cells. Environ Exp Bot 113:28–39
Rodríguez-Serrano M, Romero-Puertas MC, Sparkes I, Hawes C, del Río LA, Sandalio LM (2009) Peroxisome dynamics in Arabidopsis plants under oxidative stress induced by cadmium. Free Radic Biol Med 47:1632–1639
Tamás L, Bočová B, Huttová J, Mistrík I, Zelinová V (2017) Depletion of extracellular calcium increases cadmium toxicity in barley root tip via enhanced Cd uptake-mediated superoxide generation and cell death. Acta Physiol Plant. doi:10.1007/s11738-017-2350-8
Vaculík M, Landberg T, Greger M, Luxová M, Stoláriková M, Lux A (2012) Silicon modifies root anatomy, and uptake and subcellular distribution of cadmium in young maize plants. Ann Bot 110:433–443
Yang Y, Wei X, Lu J, You J, Wang W, Shi R (2010) Lead-induced phytotoxicity mechanism involved in seed germination and seedling growth of wheat (Triticum aestivum L.). Ecotoxicol Environ Saf 73:1982–1987
Acknowledgements
The work was supported by project IGA VFU Brno No. 321/2015/FaF (University of Veterinary and Pharmaceutical Sciences Brno). The authors thank Dr. Josef Hedbavny for the quantification of Cd.
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Communicated by O. Ferrarese-Filho.
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Kováčik, J., Babula, P. Fluorescence microscopy as a tool for visualization of metal-induced oxidative stress in plants. Acta Physiol Plant 39, 157 (2017). https://doi.org/10.1007/s11738-017-2455-0
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DOI: https://doi.org/10.1007/s11738-017-2455-0