NADPH oxidase is involved in regulation of gene expression and ROS overproduction in soybean (Glycine max L.) seedlings exposed to cadmium

Jagna Chmielowska-Bąk, Magdalena Arasimowicz-Jelonek, Karolina Izbiańska, Marina Frontasyeva, Inga Zinicovscaia, Carolina Guiance-Varela, Joanna Deckert


Cadmium-induced oxidative burst is partially mediated by NADPH oxidase. The aim of the present research was to evaluate the role of NADPH oxidase in soybeans’ response to short-term cadmium stress. The application of an NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), affected expression of two Cd-inducible genes, encoding DOF1 and MYBZ2 transcription factors. This effect was observed after 3 h of treatment. Interestingly, Cd-dependent increases in NADPH oxidase activity occurred only after a period of time ranging from 6 and 24 h of stress. Stimulation of the enzyme correlated in time with a significant accumulation of reactive oxygen species (ROS). Further analysis revealed that pharmacological inhibition of NADPH oxidase activity during 24 h of Cd stress does not affect Cd uptake, seedling growth, or the level of lipid peroxidation. The role of NADPH oxidase in the response of soybean seedlings to short-term Cd exposure is discussed.


cadmium; soybean; NADPH oxidase; ROS signaling; MYB transcription factor; DOF1 transcription factor

Full Text:



Khan S, Khan MA, Rehman S. Lead and cadmium contamination of different roadside soils and plants in Peshawar City, Pakistan. Pedosphere. 2011;21:351–357.

Wang L, Cui X, Cheng H, Chen F, Wang J, Zhao X, et al. A review of soil cadmium contamination in China including a health risk assessment. Environ Sci Pollut Res Int. 2015;22:16441–16452.

Gallego SM, Pena LB, Barcia RA, Azpilicueta CE, Iannone MF, Rosales EP, et al. Unravelling cadmium toxicity and tolerance in plants: insights into regulatory mechanisms. Environ Exp Bot. 2012;83:33–46.

Tran TA, Popova LP. Functions and toxicity of cadmium in plants: recent advances and future prospects. Turk J Botany. 2013;37:1–13.

Chmielowska-Bąk J, Deckert J. A common response to common danger? Comparison of animal and plant signaling pathways involved in cadmium sensing. J Cell Commun Signal. 2012;6:191–204.

Garnier L, Simon-Plas F, Thuleau P, Agnel JP, Blein JP, Ranjeva R, Montillet JL. Cadmium affects tobacco cells by a series of three waves of reactive oxygen species that contribute to cytotoxicity. Plant Cell Environ. 2006;29:1956–1569.

Arasimowicz-Jelonek M, Floryszak-Wieczorek J, Deckert J, Rucińska-Sobkowiak R, Gzyl J, Pawlak-Sprada S, et al. Nitric oxide implication in cadmium-induced programmed cell death in roots and signaling response of yellow lupine plants. Plant Physiol Biochem. 2012;58:124–134.

Peréz-Chaca MV, Rodrígues-Serrano M, Molina AS, Pedranzani HE, Zirulnik F, Sandalio LM, et al. Cadmium induces two waves of reactive oxygen species in Glycine max (L.) roots. Plant Cell Environ. 2014;37:1672–1687.

Smiri M. Effect of cadmium on germination, growth, redox and oxidative properties in Pisum sativa seeds. Journal of Environemntal Chemistry and Ecotoxicology. 2011;3:52–59.

Yeh CM, Chien PS, Huang J. Distinct signaling pathways for induction of MAP kinase activities by cadmium and copper in rice roots. J Exp Bot. 2007;58:659–671.

Cho SC, Chao YY, Hong CY, Kao CH. The role of hydrogen peroxide in cadmium-inhibited root growth of rice seedlings. Plant Growth Regul. 2012;66:27–35.

Maksymiec W, Krupa Z. The effect of short-term exposition to Cd, excess Cu ions and jasmonate on oxidative stress appearing in Arabidopsis thaliana. Environ Exp Bot. 2006;57:187–194.

Olmos E, Martínez-Solano JR, Piqueras A, Hellín E. Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line). J Exp Bot. 2003;54:291–301.

Rodríguez-Serrano M, Romero-Puertas MC, Zabalza A, Corpas F, Gómez M, del Río LA, et al. Cadmium effect on oxidative metabolism of pea (Pisum sativum L.) roots. Imaging of reactive oxygen species and nitric oxide accumulation in vivo. Plant Cell Environ. 2006;29:1532–1544.

Das K, Roychoudhury A. Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Front Environ Sci. 2014;2:53.

Rio LA. ROS and RNS in plant physiology: an overview. J Exp Bot. 2015;66:2827–2837.

Ge W, Jiao YQ, Sun BL, Qin R, Jiang WS, Liu DH. Cadmium-mediated oxidative stress and ultrastructure changes in root cells of poplar cultivars. S Afr J Bot. 2012;83:98–108.

Gill SS, Khan NA, Tuteja N. Cadmium at high dose perturbs growth, photosynthesis and nitrogen metabolism while at low dose it up regulates sulfur assimilation and antioxidant machinery in garden cress (Lepidum sativum L.). Plant Sci. 2012;182:112–120.

Monteiro C, Santos C, Pinho S, Oliveira H, Pedrosa T, Dias CD. Cadmium-induced cyto- and genotoxicity are organ-dependent in lettuce. Chem Res Toxicol. 2012;25:1423–1434.

Xu Q, Min H, Cai S, Fu Y, Sha S, Xie K, et al. Subcellular distribution and toxicity of cadmium in Potamogeton crispus L. Chemosphere. 2012;89:114–120.

Gonçalves JF, Becker AG, Cargnelutti D, Tabaldi LA, Pereira LB, Battisti V, et al. Cadmium toxicity causes oxidative stress and induces response of the antioxidant system in cucumber seedlings. Braz J Plant Physiol. 2007;19:223–232.

Pena LB, Pasquini LA, Tomaro ML, Gallego SM. Proteolytic system in sunflower (Heliathus annus L.) leaves under cadmium stress. Plant Sci. 2006;171:531–537.

Pena LB, Pasquini LA, Tomaro ML, Gallego SM. 20S Proteosome and accumulation of oxidized and ubiquitinated proteins in maize leaves subjected to cadmium stress. Phytochemistry. 2007;68:1139–1146.

Pena LB, Barcia RA, Azpilicueta CE, Méndez AAE, Gallego SM. Oxidative post translation modifications of proteins related to cell cycle are involved in cadmium toxicity in wheat seedlings. Plant Sci. 2012;196:1–7.

Romero-Puertas MC, Palma JM, Gómez M, del Río LA, Sandalio LM. Cadmium causes the oxidative modification of proteins in pea plants. Plant Cell Environ. 2002;25:677–686.

Choudhury S, Panda P, Sahoo L, Panda SK. Reactive oxygen species signaling in plants under abiotic stress. Plant Signal Behav. 2013;8:e23681.

Kreslavski VD, Los DA, Allakhverdiev SI, Kuznetsov VlV. Signaling role of reactive oxygen species in plants under stress. Russ J Plant Physiol. 2012;59:141–154.

Neill S, Desikan R, Hancock J. Hydrogen peroxide signaling. Curr Opin Plant Biol. 2002;5:388–395.

Wrzaczek M, Brosché M, Kangasjärvi J. ROS signaling loops – production, perception and regulation. Curr Opin Plant Biol. 2013;16:575–582.

Waszczak C, Akter S, Jacques S, Huang J, Messens J, Breusegem F. Oxidative post-translational modifications of cysteine residues in plant signal transduction. J Exp Bot. 2015;66:2923–2934.

Chi YH, Paeng SK, Kim MJ, Hwang GY, Melencion SMB, Oh HT, et al. Redox-dependent functional switching of plant proteins accompanying with their structural changes. Front Plant Sci. 2013;4:277.

Kopczewski T, Kuźniak E. Redox signals as a language of inter-organellar communication in plant cells. Cent Eur J Biol 2013;8:1153–1163.

Chmielowska-Bąk J, Izbiańska K, Deckert J. Products of lipid, protein and RNA oxidation as signals and regulators of gene expression in plants. Front Plant Sci. 2015;6:405.

Mǿller MI, Sweetlove LJ. ROS signalling – specifity is required. Trends Plant Sci. 2010;15:370–374.

Kung CP, Wu YR, Chuang HW. Expression of a dye-decolorizing peroxidase results in hypersensitive response to cadmium stress through reduction the ROS signal in Arabidopsis. Environ Exp Bot. 2014;101:47–55.

Małecka A, Kutrowska A, Piechalak A, Tomaszewksa B. High peroxide level may be a characteristic trait of a hyperaccumulator. Water Air Soil Pollut. 2015;226:84.

Chmielowska-Bąk J, Lefèvre I, Lutts S, Deckert J. Short term signaling responses in roots of young soybean seedlings exposed to cadmium stress. J Plant Physiol. 2013;170(18): 1585–1594.

Asgher M, Khan MIR, Anjum NA, Khan NA. Minimising toxicity of cadmium in plants – role of plant growth regulators. Protoplasma. 2015;252:399–413.

Schellingen K, van den Straeten D, Vandebussche F, Prinsen E, Remens T, Vangronsveld J, et al. Cadmium-induced ethylene production and response in Arabidopsis thaliana rely on ACS2 and ACS6 gene expression. BMC Plan Biol. 2014;14:214.

Jonak C, Nakagami H, Hirt H. Heavy metal stress. Activation of distinct miogen-ativated protein kinase pathways by copper and cadmium. Plant Physiol. 2004;136:3276–3283.

Kim JA, Agrawal GK, Rakwal R, Han KS, Kim KN, Yun CH, et al. Molecular cloning and mRNA expression analysis of novel rice (Oryza sativa L.) MAPK kinase kinase, OsEDR1, an ortholog of Arabidopsis AtEDR1, reveal its role in defense/stress signaling pathways and development. Biochem Biophys Res Commun. 2003;300:868–876.

Liu XM, Kim KE, Kim KC, Nguyen XC, Han HJ, Jung MS, et al. Cadmium activates Arabidopsis MPK3 and MPK6 via accumulation of reactive oxygen species. Phytochemistry. 2010;71:614–618.

Zhao FY, Wang K, Zhang SY, Ren J, Liu T, Wang X. Crosstalk between ABA, auxin, MAPK signaling, and the cell cycle in cadmium-stressed rice seedlings. Acta Physiol Plant. 2014;36:1879–1892.

Arasimowicz-Jelonek M, Floryszak-Wieczorek J, Gwóźdź AE. The message of nitric oxide in cadmium challenged plants. Plant Sci. 2011;181:612–620.

Ambawat S, Sharma P, Yadav NR, Yadav RC. MYB transcription factor genes as regulators for plant responses: an overview. Physiol Mol Biol Plants. 2013;19:307–321.

Yanhui C, Xiaoyuan Y, Kun H, Meihua L, Jigang L, Zhaofeng G, et al. The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol Biol. 2006;60:107–124.

Huerto-Ocampo JA, León-Galvàn MF, Ortega-Cruz LB, Barrera-Pacheco A, de León-Rodríguez A, Mendoza-Hernàndez G, et al. Water stress induces up-regulation of DOF1 and MIF1 transcription factors and down-regulation of proteins involved in secondary metabolism in amaranth roots (Amaranthus hypochondriacus L.). Plant Biol. 2010;13:472–482.

Park SH, Lim H, Hyun SJ, Yun DW, Yoon UH, Ji H, et al. Wound-inducible expression of the OsDof1 gene promotor in a Ds insertion mutant and transgenic plants. Plant Biotechnol Rep. 2014;8:305–313.

Liao Y, Zou HF, Wei W, Hao YJ, Tian AG, Huang J, et al. Soybean GmbZIP44, GmbZIP62 and GmbZIP78 genes function as negative regulators of ABA signaling and confer salt and freezing tolerance in transgenic Arabidopsis. Planta. 2008;228:225–240.

Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:2002–2007.

Zhao S, Fernald RS. Comprehensive algorithm for quantitative real-time polymerase chain reaction. J Comput Biol. 2005;12:1047–1064.

Sagi M, Fluhr R. Superoxide production by plant homologues of the gp91phox NADPH oxidase. Modulation of activity by calcium and tobacco mosaic virus infection. Plant Physiol. 2001;126:1281–1290.

Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem. 1976;72:248–254.

Doke N. Involvement of superoxide anion in the generation of hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophtora infestans and to the hyphal wall components. Physiol Plant Pathol. 1983;23:345–357.

Becan M, Aparicio-Tejo P, Irigoyen JJ, Sanchez-Diaz M. Some enzymes of hydrogen peroxide metabolism in leaves and root nodules of Medicago sativa. Plant Physiol. 1986;82:1169–1171.

Cuypers A, Smeets K, Ruytinx J, Opdenakker K, Keunen E, Remens T, et al. The cellular redox state as a modulator in cadmium and copper responses in Arabidopsis thaliana seedlings. J Plant Physiol. 2011;168:309–316.

Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB. Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley–powdery mildew interaction. Plant J. 1997;11:1187–1194.

Suzuki N, Miller G, Morales J, Shulaev V, Torres MA, Mittler R. Respiratory burst oxidases: the engines of ROS signaling. Curr Opin Plant Biol. 2011;14:691–699.

Drummond GR, Selemidis S, Griendling KK, Sobey CG. Combating oxidative stress in vascular disease: NADPH oxidase and therapeutic targets. Nat Rev Drug Discov. 2011;10:453–471.

Altenhőfer S, Radermacher KA, Kleikers PWM, Wingler K, Schmid HHHW. Evolution of NADPH oxidase inhibitors: selectivity and mechanisms for target engagement. Antioxid Redox Signal. 2015;23:406–448.

LV W, Yang L, Xu C, Shi Z, Shao J, Xian M, et al. Cadmium disturbs the balance between hydrogen peroxidase and superoxide radical by regulating endogenous hydrogen sulfide in the root tips of Brassica rapa. Front Plant Sci. 2017;8:232.

Bazin J, Langlabe N, Vincourt P, Arribat S, Balzergue S, El-Maarouf-Bouteau, et al. Targeted mRNA oxidation regulated sunflower seed dormancy alleviation during dry after-ripening. Plant Cell. 2011;23:2196–2208.

Gao F, Rampitsch C, Chitnis VR, Humphreys GD, Jordan MC, Ayele BT. Integrated analysis of seed proteome and mRNA oxidation reveals distinct post-transcriptional features regulating dormancy in wheat (Triticum aesativum L.). Plant Biotechnol J. 2013;11:921–932.

Shan X, Chang Y, Lin CL. Messenger RNA oxidation is an early event preceding cell death and causes reduced protein expression. FASEB J. 2007;21:2753–2764.

Kurai T, Wakayama M, Abiko T, Yanagisawa S, Aoki N, Ohsugi R. Introduction of the ZmDof1 gene into rice enhances carbon and nitrogen assimilation under low-nitrogen conditions. Plant Biotechnol J. 2011;9:826–837.

Yanagisawa S, Akiyama A, Kisaka H, Uchimiya H, Miwa T. Metabolic engineering with Dof1 transcription factor in plants: improved nitrogen assimilation and growth under low-nitrogen conditions. Proc Natl Acad Sci USA. 2004;101:7833–7838.

Riganti C, Gazzano E, Polimeni M, Costamagna C, Bosia A, Ghigo D. Diphenyleneiodonium inhibits cell redox metabolism and induces oxidative stress. J Biol Chem. 2004;279:47726–47731.

Du H, Yang SS, Liang Z, Feng BR, Liu L, Huang YB, et al. Genome-wide analysis of the MYB transcription factor superfamily in soybean. BMC Plant Biol. 2012;12:106.

Du H, Zhang L, Liu L, Tang XF, Yang WJ, Wu YM, et al. Biochemical and molecular characterization of plant MYB transcription factor family. Biochemistry (Mosc). 2009;74:1–11.

Zhang L, Wang Y, Sun M, Wang J, Kawabata S, Li Y. BrMYB4, a suppressor of genes for phenylpropanoid and anthocyanin biosynthesis, is down-regulated by UV-B but not by pigment-inducing sunlight in turnip cv. Tsuda. Plant Cell Physiol. 2014;55:2092–2101.

Pawlak-Sprada S, Arasimowicz-Jelonek M, Podgórska M, Deckert J. Activation of phenylpropanoid pathway in legume plants exposed to heavy metals. Part I. Effects of cadmium and lead on phenylalanine ammonia-lyase gene expression, enzyme activity and lignin content. Acta Biochim Pol. 2011;58:211–216.

Gzyl J, Rymer K, Gwóźdź EA. Differential response of antioxidant enzymes to cadmium stress in tolerant and sensitive cell line of cucumber (Cucumis sativus L.). Acta Biochim Pol. 2009;56:723–727.

Jakubowska D, Janicka-Russak M, Kabała K, Migocka M, Reda M. Modification of plasma membrane NADPH oxidase activity in cucumber seedling roots in response to cadmium stress. Plant Sci. 2015;234:50–59.

Gzyl J, Izbiańska K, Floryszak-Wieczorek J, Jelonek T, Arasimowicz-Jelonek M. Cadmium affects peroxynitrite generation and tyrosine nitration in seedling roots of soybean (Glycine max L.). Environ Exp Bot. 2016;131:155–163.

Yakimova ET, Kapchina-Toteva VM, Laarhoven LJ, Harren FM, Woltering EJ. Involvement of ethylene and lipid signaling in cadmium-induced cell death in tomato suspension cultures. Plant Physiol Biochem. 2006;44:581–589.

Gupta DK, Pena LB, Romero-Puertas MC, Hernández A, Inouhe M, Sandalio LM. NADPH oxidases differentially regulate ROS metabolism and nutrient uptake under cadmium toxicity. Plant Cell Environ. 2017;40(4):509–526.