, Volume 248, Issue 1, pp 49–68 | Cite as

Counteractive mechanism (s) of salicylic acid in response to lead toxicity in Brassica juncea (L.) Czern. cv. Varuna

  • Ashish Agnihotri
  • Praveen Gupta
  • Anuj Dwivedi
  • Chandra Shekhar Seth
Original Article


Main conclusion

Salicylic acid alleviates lead toxicity in Brassica juncea (L.) by promoting growth under non-stress and activating stress-defense mechanism (s) under lead stress conditions. It also boosts the ascorbate–glutathione cycle and thus helps in minimizing oxidative and DNA damage.

Brassica juncea plants were exposed to different concentrations (0, 500, 1000 and 2000 mg kg−1) of lead (Pb) and subsequently sprayed with 0.5 mM of salicylic acid (SA) to check for morphological and leaf gas exchange parameters like transpiration rate (E), stomatal conductance (GH2O), net photosynthetic rate (A) and maximum quantum yield of PS II (Fv/Fm). Leaf epidermis by scanning electron microscopy (SEM), enzymatic and non-enzymatic components of ascorbate–glutathione (AsA–GSH) cycle, DNA damage by comet assay, lipid peroxidation and endogenous SA quantification by HPLC were analyzed. Lead accumulation in root, shoot and its sub-cellular distribution ratio (SDR) and localization was also determined using atomic absorption spectroscopy (AAS) and rhodizonate-dye staining method, respectively. Results revealed that notable amount of Pb was accumulated in root and shoot in dose-dependent manner which significantly (P ≤ 0.05) posed the toxicity on the majority of morphological parameters, structural integrity of epidermal and guard cells, photosynthetic pigments, malondialdehyde (MDA) and H2O2 content. Notable decrease in leaf gas exchange parameters, Fv/Fm, poor performance of AsA–GSH cycle and striking amount of DNA damage, was found as well. However, SA revoked Pb toxicity to a great extent by promoting growth, chlorophyll content, improving the A, Fv/Fm, boosting the overall performance of AsA–GSH cycle and by lessening the DNA damage.


Ascorbate–glutathione cycle Brassica juncea Chlorophyll fluorescence DNA damage Lead Oxidative stress Photosynthetic rate Salicylic acid Sub-cellular distribution ratio 



Net photosynthetic rate


Reduced ascorbate


Transpiration rate


Stomatal conductance


Oxidized glutathione


Heavy metal


Salicylic acid



The present work is supported by Research and Development Grant of University of Delhi, India, and DU-DST Purse Grant of Department of Science and Technology, New Delhi, India. Mr. Ashish Agnihotri is deeply indebted to Council of Scientific and Industrial Research (CSIR), New Delhi, India, for awarding the Senior Research Fellowship (SRF). The technical staffs of Central Instrumentation Facility, Department of Botany, are also acknowledged for their support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Acosta-Motos JR, Ortuño MF, Álvarez S, López-Climent MF, Gómez-Cadenas A, Sánchez-Blanco MJ (2016) Changes in growth, physiological parameters and the hormonal status of Myrtus communis L. plants irrigated with water with different chemical compositions. J Plant Physiol 191:12–21CrossRefPubMedGoogle Scholar
  2. Alexieva V, Sergiev I, Mapelli S, Karanov E (2001) The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant, Cell Environ 24:1337–1344CrossRefGoogle Scholar
  3. APHA, AWWA, WEF (2005) Standard methods for the examination of water and wastewater, 21st edn. APHA, AWWA, WEF, Washington, DCGoogle Scholar
  4. Arfan M, Athar HR, Ashraf M (2007) Does exogenous application of salicylic acid through the rooting medium modulate growth and photosynthetic capacity in two differently adapted spring wheat cultivars under salt stress? J Plant Physiol 164:685–694CrossRefPubMedGoogle Scholar
  5. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24:1–15CrossRefPubMedPubMedCentralGoogle Scholar
  6. Baumann HA, Morrison L, Stengel DB (2009) Metal accumulation and toxicity measured by PAM-chlorophyll fluorescence in seven species of marine macroalgae. Ecotoxicol Environ Saf 72:1063–1075CrossRefPubMedGoogle Scholar
  7. Bazzaz FA, Carlson RW, Rolfe GL (1974) The effect of heavy metals on plants: Part I. Inhibition of gas exchange in sunflower by Pb, Cd, Ni and Tl. Environ Pollut 7:241–246CrossRefGoogle Scholar
  8. Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287CrossRefPubMedPubMedCentralGoogle Scholar
  9. Begonia MT, Begonia GB, Ighoavodha M, Gilliard D (2005) Lead accumulation by tall fescue (Festuca arundinacea Schreb.) grown on a lead-contaminated soil. Int J Environ Res Public Health 2:228–233CrossRefPubMedPubMedCentralGoogle Scholar
  10. Cheng H, Jiang ZY, Liu Y, Ye ZH, Wu ML, Sun CC, Sun FL, Fei J, Wang YS (2014) Metal (Pb, Zn and Cu) uptake and tolerance by mangroves in relation to root anatomy and lignification/suberization. Tree Physiol 34:646–656CrossRefPubMedGoogle Scholar
  11. Chin DC, Hsieh CC, Lin HY, Yeh KW (2016) A low glutathione redox state couples with a decreased ascorbate redox ratio to accelerate flowering in oncidium orchid. Plant Cell Physiol 57:423–436CrossRefPubMedGoogle Scholar
  12. Choudhury FK, Rivero RM, Blumwald E, Mittler R (2016) Reactive oxygen species, abiotic stress and stress combination. Plant J. PubMedCrossRefGoogle Scholar
  13. Clemens S (2006) Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 88:1707–1719CrossRefPubMedGoogle Scholar
  14. Clemens S, Ma JF (2016) Toxic heavy metal and metalloid accumulation in crop plants and foods. Annu Rev Plant Biol 67:489–512CrossRefPubMedGoogle Scholar
  15. Drążkiewicz M, Baszyński T (2005) Growth parameters and photosynthetic pigments in leaf segments of Zea mays exposed to cadmium, as related to protection mechanisms. J Plant Physiol 162:1013–1021CrossRefPubMedGoogle Scholar
  16. Eichhorn H, Klinghammer M, Becht P, Tenhaken R (2006) Isolation of a novel ABC-transporter gene from soybean induced by salicylic acid. J Exp Bot 57:2193–2201CrossRefPubMedGoogle Scholar
  17. El-Tayeb MA, El-Enany AE, Ahmed NL (2006) Salicylic acid-induced adaptive response to copper stress in sunflower (Helianthus annuus L.). Plant Growth Regul 50:191–199CrossRefGoogle Scholar
  18. Erturk FA, Ay H, Nardemir G, Agar G (2013) Molecular determination of genotoxic effects of cobalt and nickel on maize (Zea mays L.) by RAPD and protein analyses. Toxicol Ind Health 29:662–671CrossRefPubMedGoogle Scholar
  19. Fariduddin Q, Hayat S, Ahmad A (2003) Salicylic acid influences net photosynthetic rate, carboxylation efficiency, nitrate reductase activity, and seed yield in Brassica juncea. Photosynthetica 41:281–284CrossRefGoogle Scholar
  20. Fischer S, Kühnlenz T, Thieme M, Schmidt H, Clemens S (2014) Analysis of plant Pb tolerance at realistic submicromolar concentrations demonstrates the role of phytochelatin synthesis for Pb detoxification. ‎Environ. Sci Technol 48:7552–7559CrossRefGoogle Scholar
  21. Foster JG, Hess JL (1980) Responses of superoxide dismutase and glutathione reductase activities in cotton leaf tissue exposed to an atmosphere enriched in oxygen. Plant Physiol 66:482–487CrossRefPubMedPubMedCentralGoogle Scholar
  22. Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17:1866–1875CrossRefPubMedPubMedCentralGoogle Scholar
  23. Gichner T, Patková Z, Száková J, Demnerová K (2004) Cadmium induces DNA damage in tobacco roots, but no DNA damage, somatic mutations or homologous recombination in tobacco leaves. Mutat Res, Genet Toxicol Environ Mutagen 559:49–57CrossRefGoogle Scholar
  24. Gichner T, Patková Z, Száková J, Demnerová K (2006) Toxicity and DNA damage in tobacco and potato plants growing on soil polluted with heavy metals. Ecotoxicol Environ Saf 65:420–426CrossRefPubMedGoogle Scholar
  25. Glater RAB, Hernandez L Jr (1972) Lead detection in living plant tissue using a new histochemical method. J Air Pollut Control Assoc 22:463–467CrossRefPubMedGoogle Scholar
  26. Guo Q, Meng L, Mao PC, Jia YQ, Shi YJ (2013) Role of exogenous salicylic acid in alleviating cadmium-induced toxicity in Kentucky bluegrass. Biochem Syst Ecol 50:269–276CrossRefGoogle Scholar
  27. Gupta P, Srivastava S, Seth CS (2017) 24-Epibrassinolide and sodium nitroprusside alleviate the salinity stress in Brassica juncea (L.) cv. Varuna through cross talk among proline, nitrogen metabolism and abscisic acid. Plant Soil 411:483–498CrossRefGoogle Scholar
  28. Halliwell B (1987) Oxidative damage, lipid peroxidation and antioxidant protection in chloroplasts. Chem Phys Lipids 44:327–340CrossRefGoogle Scholar
  29. Han Y, Zhang L, Yang Y, Yuan H, Zhao J, Gu J, Huang S (2016) Pb uptake and toxicity to Iris halophila tested on Pb mine tailing materials. Environ Pollut 214:510–516CrossRefPubMedGoogle Scholar
  30. He B, Yang XE, Ni WZ, Wei YZ, Ye HB (2003) Pb uptake, accumulation, subcellular distribution in a Pb-accumulating ecotype of Sedum alfredii (Hance). J Zhejiang Univ 4:474–479CrossRefGoogle Scholar
  31. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198CrossRefPubMedGoogle Scholar
  32. Hiscox JD, Israelstam GF (1979) A method for the extraction of chlorophyll from leaf tissue without maceration. Can J Bot 57:1332–1334CrossRefGoogle Scholar
  33. Hossain MA, Nakano Y, Asada K (1984) Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell Physiol 25:385–395Google Scholar
  34. Hu ZB, Cools T, De Veylder L (2016) Mechanisms used by plants to cope with DNA damage. Annu Rev Plant Biol 67:439–462CrossRefPubMedGoogle Scholar
  35. Järup L (2003) Hazards of heavy metal contamination. Br Med Bull 68:167–182CrossRefPubMedGoogle Scholar
  36. Kersten G, Majestic B, Quigley M (2017) Phytoremediation of cadmium and lead-polluted watersheds. Ecotoxicol Environ Saf 137:225–232CrossRefPubMedGoogle Scholar
  37. Khan MI, Fatma M, Per TS, Anjum NA, Khan NA (2015) Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants. Front Plant Sci 6:462PubMedPubMedCentralGoogle Scholar
  38. Kong J, Dong Y, Xu L, Liu S, Bai X (2014) Effects of foliar application of salicylic acid and nitric oxide in alleviating iron deficiency induced chlorosis of Arachis hypogaea L. Bot Stud 55:9. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Li T, Hu Y, Du X, Tang H, Shen C, Wu J (2014) Salicylic acid alleviates the adverse effects of salt stress in Torreya grandis cv. Merrillii seedlings by activating photosynthesis and enhancing antioxidant systems. PLoS ONE 9(10):e109492CrossRefPubMedPubMedCentralGoogle Scholar
  40. Lozano-Rodriguez E, Hernandez LE, Bonay P, Carpena-Ruiz RO (1997) Distribution of cadmium in shoot and root tissues of maize and pea plants: physiological disturbances. J Exp Bot 48:123–128CrossRefGoogle Scholar
  41. MacFarlane GR, Burchett MD (2002) Toxicity, growth and accumulation relationships of copper, lead and zinc in the grey mangrove Avicennia marina (Forsk.) Vierh. Mar Environ Res 54:65–84CrossRefPubMedGoogle Scholar
  42. Martel AB, Qaderi MM (2016) Does salicylic acid mitigate the adverse effects of temperature and ultraviolet-B radiation on pea (Pisum sativum) plants? Environ Exp Bot 122:39–48CrossRefGoogle Scholar
  43. Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a practical guide. J Exp Bot 51:659–668CrossRefPubMedPubMedCentralGoogle Scholar
  44. Miura K, Tada Y (2014) Regulation of water, salinity, and cold stress responses by salicylic acid. Front Plant Sci 5:4CrossRefPubMedPubMedCentralGoogle Scholar
  45. Mostofa MG, Fujita M (2013) Salicylic acid alleviates copper toxicity in rice (Oryza sativa L.) seedlings by up-regulating antioxidative and glyoxalase systems. Ecotoxicology 22:959–973CrossRefPubMedGoogle Scholar
  46. Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880Google Scholar
  47. Pancheva TV, Popova LP (1998) Effect of salicylic acid on the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase in barley leaves. J Plant Physiol 152:381–386CrossRefGoogle Scholar
  48. Pancheva TV, Popova LP, Uzunova AN (1996) Effects of salicylic acid on growth and photosynthesis in barley plants. J Plant Physiol 149:57–63CrossRefGoogle Scholar
  49. Peleg Z, Blumwald E (2011) Hormone balance and abiotic stress tolerance in crop plants. Curr Opin Plant Biol 3:290–295CrossRefGoogle Scholar
  50. Pilon-Smits E (2005) Phytoremediation. Annu Rev Plant Biol 56:15–39CrossRefPubMedGoogle Scholar
  51. Rai VK, Sharma SS, Sharma S (1986) Reversal of ABA-induced stomatal closure by phenolic compounds. J Exp Bot 37:129–134CrossRefGoogle Scholar
  52. Rai R, Agrawal M, Agrawal SB (2016) Impact of heavy metals on physiological processes of plants: With special reference to photosynthetic system. Plant responses to xenobiotics. Springer, Singapore, pp 127–140Google Scholar
  53. Rajasekaran LR, Blake TJ (1999) New plant growth regulators protect photosynthesis and enhance growth under drought of jack pine seedlings. Plant Growth Regul 18:175–181CrossRefGoogle Scholar
  54. Reis GSM, de Almeida AAF, de Almeida NM, de Castro AV, Mangabeira PAO, Pirovani CP (2015) Molecular, biochemical and ultrastructural changes induced by Pb toxicity in seedlings of Theobroma cacao L. PLoS ONE 10:e0129696CrossRefPubMedPubMedCentralGoogle Scholar
  55. Rivas-San Vicente M, Plasencia J (2011) Salicylic acid beyond defense: its role in plant growth and development. J Exp Bot 62:3321–3338CrossRefPubMedGoogle Scholar
  56. Rüdiger W (2002) Biosynthesis of chlorophyll b and the chlorophyll cycle. Photosynth Res 74:187–193CrossRefPubMedGoogle Scholar
  57. Salt DE, Blaylock M, Kumar NP, Dushenkov V, Ensley BD, Chet I, Raskin I (1995) Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Biotechnology 13:468–474PubMedGoogle Scholar
  58. Scalabrin E, Radaelli M, Capodaglio G (2016) Simultaneous determination of shikimic acid, salicylic acid and jasmonic acid in wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses. Plant Physiol Biochem 103:53–60CrossRefPubMedGoogle Scholar
  59. Sharma SS, Dietz KJ, Mimura T (2016) Vacuolar compartmentalization as indispensable component of heavy metal detoxification in plants. Plant, Cell Environ 39:1112–1126. CrossRefGoogle Scholar
  60. Singh S, Sinha S (2005) Accumulation of metals and its effects in Brassica juncea (L.) Czern. (cv. Rohini) grown on various amendments of tannery waste. Ecotoxicol Environ Saf 62:118–127CrossRefPubMedGoogle Scholar
  61. Singh S, Bhatia A, Tomer R, Kumar V, Singh B, Singh SD (2013) Synergistic action of tropospheric ozone and carbon dioxide on yield and nutritional quality of Indian mustard (Brassica juncea (L.) Czern.). Environ Model Assess 8:6517–6529CrossRefGoogle Scholar
  62. Stevens J, Senaratna T, Sivasithamparam K (2006) Salicylic acid induces salinity tolerance in tomato (Lycopersicon esculentum cv. Roma): associated changes in gas exchange, water relations and membrane stabilisation. Plant Growth Regul 49:77–83Google Scholar
  63. Sytar O, Kumar A, Latowski D, Kuczynska P, Strzałka K, Prasad MNV (2013) Heavy metal-induced oxidative damage, defense reactions, and detoxification mechanisms in plants. Acta Physiol Plant 35:985–999CrossRefGoogle Scholar
  64. Vanacker H, Lu H, Rate DN, Greenberg JT (2001) A role for salicylic acid and NPR1 in regulating cell growth in Arabidopsis. Plant J 28:209–216CrossRefPubMedGoogle Scholar
  65. Venema DP, Hollman PC, Janssen KP, Katan MB (1996) Determination of acetylsalicylic acid and salicylic acid in foods, using HPLC with fluorescence detection. J Agric Food Chem 44:1762–1767CrossRefGoogle Scholar
  66. Wang P, Zhang S, Wang C, Lu J (2012) Effects of Pb on the oxidative stress and antioxidant response in a Pb bioaccumulator plant Vallisneria natans. Ecotoxicol Environ Saf 78:28–34CrossRefPubMedGoogle Scholar
  67. Wu QS, Xia RX, Zou YN (2006) Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress. J Plant Physiol 163:1101–1110CrossRefPubMedGoogle Scholar
  68. Yang ZM, Wang J, Wang SH, Xu LL (2003) Salicylic acid-induced aluminum tolerance by modulation of citrate efflux from roots of Cassia tora L. Planta 217:168–174PubMedGoogle Scholar
  69. Yang Y, Han X, Liang Y, Ghosh A, Chen J, Tang M (2015) The combined effects of arbuscular mycorrhizal fungi (AMF) and lead (Pb) stress on Pb accumulation, plant growth parameters, photosynthesis, and antioxidant enzymes in Robinia pseudoacacia L. PLoS ONE 10(12):e0145726CrossRefPubMedPubMedCentralGoogle Scholar
  70. Yuan L, Shu S, Sun J, Guo S, Tezuka T (2012) Effects of 24-epibrassinolide on the photosynthetic characteristics, antioxidant system, and chloroplast ultrastructure in Cucumis sativus L. under Ca (NO3)2 stress. Photosynth Res 112:205–214CrossRefPubMedGoogle Scholar
  71. Zha TS, Wu YJ, Jia X, Zhang MY, Bai YJ, Liu P, Ma JY, Bourque CPA, Peltola H (2017) Diurnal response of effective quantum yield of PSII photochemistry to irradiance as an indicator of photosynthetic acclimation to stressed environments revealed in a xerophytic species. Ecol Indic 74:191–197CrossRefGoogle Scholar
  72. Zhong B, Chen J, Shafi M, Guo J, Wang Y, Wu J, Ye Z, He L, Liu D (2017) Effect of lead (Pb) on antioxidation system and accumulation ability of Moso bamboo (Phyllostachys pubescens). Ecotoxicol Environ Saf 138:71–77CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Ashish Agnihotri
    • 1
  • Praveen Gupta
    • 1
  • Anuj Dwivedi
    • 1
    • 2
  • Chandra Shekhar Seth
    • 1
  1. 1.Department of BotanyUniversity of DelhiDelhiIndia
  2. 2.National Institute of Plant Genome ResearchNew DelhiIndia

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