Seed Pre-treatment with Polyhydroxy Fullerene Nanoparticles Confer Salt Tolerance in Wheat Through Upregulation of H2O2 Neutralizing Enzymes and Phosphorus Uptake

  • Fahad ShafiqEmail author
  • Muhammad Iqbal
  • Muhammad Ali
  • Muhammad Arslan Ashraf
Original Paper


Polyhydroxy fullerenes nanoparticles (PHF) are regarded as free radical sponges. Can they mitigate oxidative stress and induce tolerance in plants exposed to salinity? The influence of PHF seed pre-treatment on growth and biochemical attributes of NaCl-stressed wheat is studied. Wheat seeds (cv. Ujala) were pre-treated with control, hydro-priming, 10, 40, 80, and 120 nM PHF doses for 10 h and grown in sand-filled pots under control (0 mM NaCl) and salinity (150 mM NaCl) provided through nutrient solution. Salinity markedly decreased root and shoot growth attributes consistent with the reduction in the chlorophyll contents, whereas it increased the antioxidant activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) enzymes. Plants exposed to salinity exhibited increase in malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents which indicated oxidative stress. Further, salinity triggered rise in Na+ uptake while decreased in K+ and Ca2+ contents both in the root and shoot. By contrast, wheat seedlings grown from PHF-treated seeds exhibited recovery in root and shoot growth under salinity. This recovery was linked with lower levels of MDA and H2O2 contents and higher antioxidant activities of CAT, POD, and APX enzymes under salinity stress. The PHF-treated plants had higher chlorophyll, free amino acids, ascorbic acid, and soluble sugars. Moreover, PHF seed pre-treatment resulted in higher K+ and P contents in the root while higher P contents in the shoot. Above all, PHF application mitigated adverse effects of salinity and promoted early seedling growth and establishment in wheat.


Antioxidants Nanoparticles Oxidative stress Polyhydroxy fullerene Salinity Wheat 


Author Contributions

All the authors contributed equally to this manuscript.

Funding Information

The study was partially funded by Higher Education Commission (HEC), Islamabad, Pakistan through Project grants no. 20-1522/R&D/09 and 20-1523/R&D/10 to Prof. Dr. Muhammad Iqbal.

Compliance with Ethical Standards

Conflict of Interests

The authors declare that they have no competing interests.

Supplementary material

42729_2019_73_MOESM1_ESM.docx (47 kb)
ESM 1 (DOCX 46 kb)


  1. Aebi H (1984) Catalase in vitro (In: L. Pac). Academic Press, OrlandoGoogle Scholar
  2. Alemán F, Nieves-Cordones M, Martínez V, Rubio F (2009) Potassium/sodium steady-state homeostasis in Thellungiella halophila and Arabidopsis thaliana under long-term salinity conditions. Plant Sci 176:768–774Google Scholar
  3. Andrievsky G, Klochkov V, Derevyanchenko L (2005) Is the C60 fullerene molecule toxic?! Fuller Nanotub Car N 13(4):363–376Google Scholar
  4. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399Google Scholar
  5. Arnon DI (1949) Copper enzymes in isolated chlorlasts: polyphenoloxidase in Beta vulgaris. Plant Physiol 24(1):1–15Google Scholar
  6. Assemi S, Tadjiki S, Donose BC, Nguyen AV, Miller JD (2010) Aggregation of fullerol C60(OH)24 nanoparticles as revealed using flow field-flow fractionation and atomic force microscopy. Langmuir 26(20):16063–16070Google Scholar
  7. Barragán V, Leidi EO, Andrés Z, Rubio L, De Luca A, Fernández JA, Cubero B, Pardo JM (2012) Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis. Plant Cell 24:1127–1142Google Scholar
  8. Barton CJ (1948) Photometric analysis of phosphate rock. Anal Chem 20(11):1068–1073Google Scholar
  9. Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44(1):276–287Google Scholar
  10. Borišev M, Borišev I Župunski M, Arsenov D, Pajević S, Ćurčić Ž, Vasin J, Djordjevic A (2016) Drought impact is alleviated in sugar beets (Beta vulgaris L.) by foliar application of fullerenol nanoparticles. PLoS One 11(11):263 1-20Google Scholar
  11. 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(1–2):248–254Google Scholar
  12. Bray HG, Thorpe WV (1954) Analysis of phenolic compounds of interest in metabolism. Methods Biochem Anal 1:27–52Google Scholar
  13. Cavalcanti FR, Lima JPMS, Ferreira-Silva SL, Viegas RA, Silveira JAG (2007) Roots and leaves display contrasting oxidative response during salt stress and recovery in cowpea. J Plant Physiol 164:591–600Google Scholar
  14. Chance B, Maehly AC (1955) Assay of catalases and peroxidases. Methods Enzymol 2:764–775Google Scholar
  15. Colmer TD, Munns R, Flowers TJ (2006) Improving salt tolerance of wheat and barley: future prospects. Aust J Exp Agric 45(11):425–1443Google Scholar
  16. Cuin TA, Bose J, Stefano G, Jha D, Tester M, Mancuso S, Shabala S (2011) Assessing the role of root plasma membrane and tonoplast Na+/H+ exchanger in salinity tolerance in wheat: in planta quantification methods. Plant Cell Environ 34:947–961Google Scholar
  17. Cuin TA, Tian Y, Betts SA, Chalmandrier R, Shabala S (2009) Ionic relations and osmotic adjustment in durum and bread wheat under saline conditions. Funct Plant Biol 36:1110–1119Google Scholar
  18. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28(3):350–431Google Scholar
  19. Flowers TJ, Colmer TD (2008) Salinity tolerance in halophytes. New Phytol 179:945–963Google Scholar
  20. Foley S, Crowley C, Smaihi M, Bonfils C, Erlanger BF, Seta P, Larroque C (2002) Cellular localisation of a water-soluble fullerene derivative. Biochem Biophys Res Commun 294(1):116–119Google Scholar
  21. Gao J, Wang Y, Folta KM, Krishna V, Bai W, Indeglia P, Georgieva A, Nakamura H, Koopman B, Moudgil B (2011) Polyhydroxy fullerenes (fullerols or fullerenols): beneficial effects on growth and lifespan in diverse biological models. PLoS One 6(5):1–8Google Scholar
  22. Geilfus CM (2018) Chloride: from nutrient to toxicant. Plant Cell Physiol 59(5):877–886Google Scholar
  23. González-Pérez L, Páez-Watson T, Álvarez-Suarez JM, Obando-Rojas MC, Bonifaz-Arcos E, Viteri G, Rivas-Romero F, Tejera E, Rogers HJ, Cabrera JC (2018) Application of exogenous xyloglucan oligosaccharides affects molecular responses to salt stress in Arabidopsis thaliana seedlings. J Soil Sci Plant Nutr 18(4):1187–1205Google Scholar
  24. Hamilton PB, Van Slyke DD (1943) The gasometric determination of free amino acids in blood filtrates by the ninhydrin-carbon dioxide method. J Biol Chem 150(1):231–250Google Scholar
  25. Hasegawa PM (2013) Sodium (Na+) homeostasis and salt tolerance of plants. Environ Exp Bot 92:19–31Google Scholar
  26. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Biol 51:463–499Google Scholar
  27. Hauser F, Horie T (2010) A conserved primary salt tolerance mechanism mediated by HKT transporters: a mechanism for sodium exclusion and maintenance of high K+/Na+ ratio in leaves during salinity stress. Plant Cell Environ 33:552–565Google Scholar
  28. Iqbal M, Ashraf M (2013) Gibberellic acid mediated induction of salt tolerance in wheat plants: growth, ionic partitioning, photosynthesis, yield and hormonal homeostasis. Environ Exp Bot 86:76–85Google Scholar
  29. Kirk JT, Allen RL (1965) Dependence of chloroplast pigment synthesis on protein synthesis: effect of actidione. Biochem Biophys Res Commun 21(6):523–530Google Scholar
  30. Kole C, Kole P, Randunu KM, Choudhary P, Podila R, Ke PC, Rao AM, Marcus RK (2013) Nanobiotechnology can boost crop production and quality: first evidence from increased plant biomass, fruit yield and phytomedicine content in bitter melon (Momordica charantia). BMC Biotechnol 13(1):37Google Scholar
  31. Läuchli A, James RA, Huang CX, McCully M, Munns R (2008) Cell-specific localization of Na+ in roots of durum wheat and possible control points for salt exclusion. Plant Cell Environ 31:1565–1574Google Scholar
  32. Liu F, Xiong F, Fan Y, Li J, Wang H, Xing G, Yan F, Tai F, He R (2016) Facile and scalable fabrication engineering of fullerenol nanoparticles by improved alkaline-oxidation approach and its antioxidant potential in maize. J Nanopart Res 483(18):338Google Scholar
  33. Marshner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic Press, New YorkGoogle Scholar
  34. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7(9):405–410Google Scholar
  35. Mittler R (2006) Abiotic stress, the field environment and stress combination. Trends Plant Sci 11:15–19Google Scholar
  36. Mukherjee SP, Choudhuri MA (1983) Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings. Physiol Plant 58(2):166–170Google Scholar
  37. Munns R, James RA, Gilliham M, Flowers TJ, Colmer TD (2016) Tissue tolerance: an essential but elusive trait for salt-tolerant crops. Funct Plant Biol 43(498):1103–1113Google Scholar
  38. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681Google Scholar
  39. Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22(5):867–880Google Scholar
  40. Nedjimi B (2017) Calcium application enhances plant salt tolerance: a review. In: Naeem M, Ansari A, Gill S (eds) Essential plant nutrients. Springer, ChamGoogle Scholar
  41. Panova GG, Ktitorova IN, Skobeleva OV, Sinjavina NG, Charykov NA, Semenov KN (2016) Impact of polyhydroxy fullerene (fullerol or fullerenol) on growth and biophysical characteristics of barley seedlings in favourable and stressful conditions. Plant Growth Regul 79(3):309–317Google Scholar
  42. Pardo JM, Rubio F (2011) Na+ and K+ transporters in plant signaling. In: Transporters and pumps in plant signaling. Springer, Berlin Heidelberg, pp 65–98Google Scholar
  43. Pękal A, Pyrzynska K (2014) Evaluation of aluminium complexation reaction for flavonoid content assay. Food Anal Methods 7(9):1776–1782Google Scholar
  44. Percey WJ, Shabala L, Wu Q, Su N, Breadmore MC, Guijt RM, Bose J, Shabala S (2016) Potassium retention in leaf mesophyll as an element of salinity tissue tolerance in halophytes. Plant Physiol Biochem 109:346–354Google Scholar
  45. Raza SH, Athar HR, Ashraf M, Hameed A (2007) Glycinebetaine-induced modulation of antioxidant enzymes activities and ion accumulation in two wheat cultivars differing in salt tolerance. Environ Exp Bot 60(3):368–376Google Scholar
  46. Raza SH, Ahmad MB, Ashraf MA, Shafiq F (2014) Time-course changes in growth and biochemical indices of mung bean [Vigna radiata (L.) Wilczek] genotypes under salinity. Braz J Bot 37(4):429–439Google Scholar
  47. Sachkova AS, Kovel ES, Vorobeva AA, Kudryasheva NS (2017) Antioxidant activity of fullerenols. Bioluminescent monitoring in vitro. Procedia Tech 27:230–231Google Scholar
  48. Shabala S, Shabala S, Cuin TA, Pang J, Percey W, Chen Z, Conn S, Eing C, Wegner LH (2010) Xylem ionic relations and salinity tolerance in barley. Plant J 61:839–853Google Scholar
  49. Shabala L, Zhang J, Pottosin I, Bose J, Zhu M, Fuglsang AT, Velarde-Buendia A, Massart A, Hill CB, Roessner U, Shabala S (2016) Cell-type-specific H+-ATPase activity in root tissues enables K+ retention and mediates acclimation of barley (Hordeum vulgare) to salinity stress. Plant Physiol 172(4):2445–2458Google Scholar
  50. Shabala S (2017) Signalling by potassium: another second messenger to add to the list? J Exp Bot 68(15):4003–4007Google Scholar
  51. Shabala S, Cuin T (2007) Potassium transport and plant salt tolerance. Physiol Plant 133:651–669Google Scholar
  52. Shafiq F, Raza SH, Bibi A, Khan I, Iqbal M (2018) Inluence of proline priming on antioxidative potential and ionic distribution and its relationship with salt tolerance of wheat. Cereal Res Commun 46(2):286–299Google Scholar
  53. Sheldon AR, Dalal RC, Kirchhof G, Kopittke PM, Menzies NW (2017) The effect of salinity on plant-available water. Plant Soil 418(1-2):477–491Google Scholar
  54. Shu S, Guo R, Sun J, Yuan Y (2012) Effects of salt stress on the structure and function of the photosynthetic apparatus in Cucumis sativus and its protection by exogenous putrescine. Physiol Plant 146:285–296Google Scholar
  55. Taiz L, Zeiger E (2010) Plant physiology, fifth edn. Sinauer Associates, SunderlandGoogle Scholar
  56. Tavakkoli E, Fatehi F, Coventry S, Rengasamy P, McDonald GK (2010) Additive effects of Na+ and Cl ions on barley growth under salinity stress. J Exp Bot 62:2189–2203Google Scholar
  57. Teakle NL, Tyerman SD (2010) Mechanisms of Cl transport contributing to salt tolerance. Plant Cell Environ 33:566–589Google Scholar
  58. Tedeschi A, Zong L, Huang CH, Vitale L, Volpe MG, Xue X (2017) Effect of salinity on growth parameters, soil water potential and ion composition in Cucumis melo cv. Huanghemi in north‐western China. J Agron Crop Sci 203(1): 41–55Google Scholar
  59. Torbaghan ME, Lakzian A, Astaraei AR, Fotovat A, Besharati H (2017) Salt and alkali stresses reduction in wheat by plant growth promoting haloalkaliphilic bacteria. J Soil Sci Plant Nutr 17(4):1058–1087Google Scholar
  60. Velikova V, Yordanov I, Edreva A (2000) Oxidative stress and some antioxidant systems in acid rain-treated bean plants: Protective role of exogenous polyamines. Plant Sci 151(1):59–66Google Scholar
  61. Wang C, Zhang H, Ruan L, Chen L, Li H, Chang XL, Zhang X, Yang ST (2016) Bioaccumulation of 13C-fullerenol nanomaterials in wheat. Environ Sci Nano 3(4):799–805Google Scholar
  62. Wolf B (1982) A comprehensive system of leaf analyses and its use for diagnosing crop nutrient status. Commun Soil Sci Plant Anal 13(12):1035–1059Google Scholar
  63. Xiong JL, Li J, Wang HC, Zhang CL, Naeem MS (2018) Fullerol improves seed germination, biomass accumulation, photosynthesis and antioxidant system in Brassica napus L. under water stress. Plant Physiol Biochem 129:130–140Google Scholar
  64. Zhang WD, Wang P, Bao Z, Ma Q, Duan LJ, Bao AK, Zhang JL, Wang SM (2017) SOS1, HKT1; 5, and NHX1 synergistically modulate Na+ homeostasis in the halophytic grass Puccinellia tenuiflora. Front Plant Sci 8:576Google Scholar
  65. Zhu J (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247–273Google Scholar

Copyright information

© Sociedad Chilena de la Ciencia del Suelo 2019

Authors and Affiliations

  1. 1.Department of BotanyGovernment College University FaisalabadFaisalabadPakistan
  2. 2.Department of Bioinformatics and BiotechnologyGovernment College University FaisalabadFaisalabadPakistan
  3. 3.Faculty of Animal SciencesQuaid-i-Azam UniversityIslamabadPakistan

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