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Effect of cadmium stress on inductive enzymatic and nonenzymatic responses of ROS and sugar metabolism in multiple shoot cultures of Ashwagandha (Withania somnifera Dunal)

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Abstract

Withania somnifera is one of the most important medicinal plant and is credited with various pharmacological activities. In this study, in vitro multiple shoot cultures were exposed to different concentrations (5–300 μM) of cadmium (Cd) as cadmium sulphate to explore its ability to accumulate the heavy metal ion and its impact on the metabolic status and adaptive responses. The results showed that supplemental exposure to Cd interfered with N, P, and K uptake creating N, P, and K deficiency at higher doses of Cd that also caused stunting of growth, chlorosis, and necrosis. The study showed that in vitro shoots could markedly accumulate Cd in a concentration-dependent manner. Enzymatic activities and isozymic pattern of catalase, ascorbate peroxidase, guaiacol peroxidase, peroxidase, glutathione-S-transferase, glutathione peroxidase, monodehydroascorbate reductase, and dehydroascorbate reductase were altered substantially under Cd exposure. Sugar metabolism was also markedly modulated under Cd stress. Various other parameters including contents of photosynthetic pigments, phenolics, tocopherol, flavonoids, reduced glutathione, nonprotein thiol, ascorbate, and proline displayed major inductive responses reflecting their protective role. The results showed that interplay of enzymatic as well as nonenzymatic responses constituted a system endeavor of tolerance of Cd accumulation and an efficient scavenging strategy of its stress implications.

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Abbreviations

ASC:

Ascorbate

APX:

Ascorbate peroxidase

BSA:

Bovine serum albumin

CAD:

Cinnamyl alcohol dehydrogenase

CAT:

Catalase

Cd:

Cadmium

DAB:

3, 3′-Diaminobenzidine

DCPIP:

2,6-Dichlorophenolindophenol

DCFH-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

DHAR:

Dehydroascorbate reductase

DHA:

Dehydroascorbate

DIA:

Diaphorase

DTNB:

5-5'-Dithiobis-(2-ntrobenzoic acid)

DTT:

Dithiothreitol

DNPH:

2,4-Dinitrophenylhydrazine

EDTA:

Ethylenediaminetetraacetic acid

EST:

Amyl esterase

FRAP:

Ferric-reducing antioxidant power property

GOT:

Glutamate oxaloacetate transaminase

G-POD:

Guaiacol peroxidase

GPX:

Glutathione peroxidase

GR:

Glutathione reductase

GSH:

Reduced glutathione

GSSG:

Oxidized glutathione

GST:

Glutathione-S-transferase

G6PDH:

Glucose-6-phosphate dehydrogenase

ICP:

Inductively coupled plasma

KPB:

Potassium phosphate buffer

MDA:

Malondialdehyde

MDHAR:

Monodehydroascorbate reductase

MS:

Murashige and Skoog

MTT:

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

NBT:

Nitroblue tetrazolium

NEM:

N-Ethylmaleimide

NPSH:

Nonprotein thiol

OAT:

Ornithine aminotransferase

PAGE:

Polyacrylamide gel electrophoresis

PAL:

Phenylalanine ammonia lyase

P5C:

Pyrroline-5-corboxylate reductase

POD:

Peroxidase

PO:

Proline oxidase

PPO:

Polyphenol oxidase

PVPP:

Polyvinylpolypyrrolidone

ROS:

Reactive oxygen species

SKDH:

Shikimate dehydrogenase

SOD:

Superoxide dismutase

TBA:

2-Thiobarbituric acid

TDW:

Triple distilled water

TBARS:

2-Thiobarbituric acid reactive substances

TCA:

Trichloroacetic acid

TPTZ:

2,4,4-Tripyridyl-2-triazine

TEMED:

N,N,N′,N′-Tetramethylethylenediamine

References

  • Ali MB, Hahn EJ, Paek KY (2007) Methyl jasmonate and salicylic acid induced oxidative stress and accumulation of phenolics in Panax ginseng bioreactor root suspension cultures. Molecules 12:607–621

    Article  CAS  PubMed  Google Scholar 

  • Bates LS, Waldran RP, Teare ID (1973) Rapid determination of free proline for water stress srudies. Plant Soil 39:205–208

    Article  CAS  Google Scholar 

  • Beltagi HS, Mohamed AA, Rashed MM (2010) Response of antioxidative enzymes to Cd stress in leaves and roots of radish (Raphanus sativus L.). Not Sci Biol 4:76–82

    Google Scholar 

  • Bernstein N, Shoresh M, Xu Y, Huang B (2010) Involvement of the plant antioxidative response in the differential growth sensitivity to salinity of leaves vs roots during cell development. Free Radic Biol Med 49:1161–1171

    Article  CAS  PubMed  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

    Article  CAS  PubMed  Google Scholar 

  • Chandrashekar S, Umesha S (2012) Induction of antioxidant enzymes associated with bacterial spot pathogenesis in tomato. Int J Food Agric Vet Sci 2:22–34

    Google Scholar 

  • Chang CC, Yang MH, Wen HM, Chern JC (2002) Estimation of total flavonoid content in Propolis by two complementary colorimetric methods. J Food Drug Anal 10:178–182

    CAS  Google Scholar 

  • Couee I, Sulmon CC, Gouesbet G, Amrani AE (2006) Involvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants. J Exp Bot 57:449–459

    Article  CAS  PubMed  Google Scholar 

  • Gill SS, Tuteja N (2011) Cd stress tolerance in crop plants probing the role of sulfur. Plant Signal Behav 6:215–222

    Article  CAS  PubMed  Google Scholar 

  • Hart JJ, Welch RM, Norvell WA, Sullivan LA (1998) Characterization of cadmium binding, uptake, and translocation in intact seedlings of bread and durum wheat cultivars. Plant Physiol 116:1413–1420

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Heath R, Packer L (1968) Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 196:385–395

    Google Scholar 

  • Hiscox JD, Israelstam GF (1979) A method for the extraction of chlorophyll from leaf tissue without maceration. Can J Bot 57:1332–1334

    Article  CAS  Google Scholar 

  • Jaleel CA, Riadh K, Gopi R, Manivannan P, Ines J, Al-Juburi HJ, Xing ZC, Bo SH, Panneerselvam R (2009) Antioxidant defense responses: physiological plasticity in higher plants under abiotic constraints. Acta Physiol Plant 31:427–436

    Google Scholar 

  • Kaur G, Singh HP, Batish DR, Kohil RK (2012) A time course assessment of changes in reactive oxygen species generation and antioxidant defense in hydroponically grown wheat in response to lead ions (Pb+2). Protoplasma 249:1091–1100

    Article  CAS  PubMed  Google Scholar 

  • Khan MA, Ahmad I, Rahman IU (2007) Effect of environmental pollution on heavy metals content of Withania somnifera. J Chin Chem Soc 54:339–343

    Google Scholar 

  • Khatun S, Ali MB, Hahn EJ, Paek KY (2008) Copper toxicity in Withania somnifera: growth and antioxidant enzymes response of in vitro grown plants. Env Exp Bot 64:279–285

    Article  CAS  Google Scholar 

  • Kivack B, Akay S (2005) Quantitative determination of a-tocopherol in Pistacia lentiscus, Pistacia lentiscus var. chia and Pistacia terebinthus by TLC-densitometry and colorimetry. Fitoterapia 76:62–66

    Article  Google Scholar 

  • Kramer U (2010) Metal hyperaccumulation in plants. Annu Rev Plant Biol 61:517–534

    Article  PubMed  Google Scholar 

  • Kováčik J, Klejdus B, Hedbavny J, Štork F, Bačkor M (2009) Comparison of cadmium and copper effect on phenolic metabolism, mineral nutrients and stress-related parameters in Matricaria chamomilla plants. Plant Soil 320:231–242

    Google Scholar 

  • Lombardi L, Sebastiani L (2005) Copper toxicity in Prunus cerasifera: growth and antioxidant enzymes responses of in vitro grown plants. Plant Sci 168:797–802

    Article  CAS  Google Scholar 

  • Madan S, Nainawatee HS, Jain RK, Chowdhury JB (1995) Proline and proline metabolizing enzymes in in-vitro selected NaCl-tolerant Brassica juncea L. under salt stress. Ann Bot 76:51–57

    Article  CAS  Google Scholar 

  • Mahmood T, Gupta KJ, Kaiser WM (2009) Cd stress stimulates nitric oxide production by wheat roots. Pak J Bot 41:1285–1290

    CAS  Google Scholar 

  • Metwally A, Safronova VI, Belimov AA, Dietz KJ (2005) Genotypic variation of the response to cadmium toxicity in Pisum sativum L. J Exp Bot 56:167–178

    CAS  PubMed  Google Scholar 

  • Mishra MK, Prakash V (2010) Response of non-enzymatic antioxidants to zinc induced stress at different pH in Glycine max L. cv. Merrill. Acad J Plant Sci 1:1–10

    Google Scholar 

  • Mittler R, Vanderauwera S, Gollery M, Breusegem FV (2004) Reactive oxygen gene network of plants. Plant Sci 9:491–498

    Article  Google Scholar 

  • Molan AL, Faraj AM, Mahdy AS (2012) Antioxidant activity and phenolic content of some medicinal plants traditionally used in northern Iraq. Phytopharmaco 2:224–233

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Namdjoyan S, Namdjoyan S, Kermanian H (2012) Induction of phytochelatin and responses of antioxidants under cadmium stress in safflower (Carthamus tinctorius) seedlings. Turk J Bot 36:495–502

    CAS  Google Scholar 

  • Nazar R, Iqbal N, Masood A, Iqbal M, Khan R, Syeed S, Khan NA (2012) Cadmium toxicity in plants and role of mineral nutrients in its alleviation. Am J Plant Sci 3:1476–1489

    Article  CAS  Google Scholar 

  • Nedjimi B, Daoud Y (2009) Cadmium accumulation in Atriplex halimus subsp. schweinfurthii and its influence on growth, proline, root hydraulic conductivity and nutrient uptake. Flora 204:316–324

    Article  Google Scholar 

  • Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 163:375–380

    Google Scholar 

  • Sabir F, Sangwan RS, Kumar R, Sangwan NS (2012) Salt stress-induced responses in growth and metabolism in callus cultures and differentiating in vitro shoots of Indian ginseng (Withania somnifera Dunal). J Plant Growth Regul 31:537–548

    Article  CAS  Google Scholar 

  • Sabir F, Mishra S, Sangwan RS, Jadaun JS, Sangwan NS (2013) Qualitative and quantitative variations in withanolides and expression of some pathway genes during different stages of morphogenesis in Withania somnifera Dunal. Protoplasma. doi:10.1007/s00709-012-0438-y

    PubMed  Google Scholar 

  • Saha P, Chatterijee P, Biswas AK (2010) NaCl pretreatment alleviates salt stress by enhancement of antioxidative defense system and osmolyte accumulation in Mungbean (Vigna radiate L. Wilczek). Indian J Exp Biol 48:593–600

    Google Scholar 

  • Sangwan NS, Yadav U, Sangwan RS (2003) Genetic diversity among elite varieties of the aromatic grasses, Cymbopogon martini. Euphytica 130:117–130

    Article  CAS  Google Scholar 

  • Sangwan RS, Chaurasiya ND, Mishra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi GN, Tuli R (2004) Phytochemical variability in commercial herbal products and preparations of Withania somnifera (ashwagandha). Curr Sci 86:461–465

    CAS  Google Scholar 

  • Sangwan RS, Chaurasiya ND, Lal P, Misra L, Uniyal GC, Tuli R, Sangwan NS (2007) Withanolide A biogeneration in in vitro shoot cultures of ashwagandha (Withania somnifera Dunal), a main medicinal plant in Ayurveda. Chem Pharm Bull 55:1371–1375

    Article  CAS  PubMed  Google Scholar 

  • Sangwan RS, Chaurasiya ND, Lal P, Mishra LN, Tuli R, Sangwan NS (2008) Root contained withanolide A is inherently de novo synthesized within roots in ashwagandha (Withania somnifera). Physiol Plant 133:278–287

    Article  CAS  PubMed  Google Scholar 

  • Schopfer P, Plachy C, Frahry G (2001) Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid. Plant Physiol 125:1591–1602

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Singh N, Luthra R (1988) Sucrose metabolism and essential oil accumulation during lemongrass (Cymbopogon flexuosus Stapf) leaf development. Plant Sci 57:127–133

    Article  CAS  Google Scholar 

  • Siregar IZ, Yunanto T (2010) The genetics of glutamate oxaloacetate transaminase (GOT) in Pinus merkusii Jungh. et de Vriese. Biodiversitas 11:5–8

    Google Scholar 

  • Tuli, R, Sangwan RS, Kumar S, Bhattacharya S, Misra LN, Mandal C, Raghubir R, Trivedi PK, Tewari SK, Mishra P, Chaturvedi P, Sangwan NS, Nair KN, Rawat AKS, Srivastava V, Srivastava RK, Ojha SK, Mehrotra S, Khajuria A, Suri KA (2009) Ashwagandha (Withania somnifera) A model Indian medicinal plant. Eds R Tuli and RS Sangwan, Publisher NMITLI-CSIR, New Delhi. ISBN No.978-93-80235-29-5

  • Tzure-Meng WU, Yi-Ting HSU, Tse-Min LEE (2009) Effects of Cd on the regulation of antioxidant enzyme activity, gene expression, and antioxidant defenses in the marine macroalga Ulva fasciata. Bot Studies 50:25–34

    Google Scholar 

  • Uchida R (2000) Essential nutrients for plant growth: nutrient functions and deficiency symptoms. College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa 31:55

  • Vajanathappa J, Badami S (2009) Antiedematogenic and free radical scavenging activity of swertiamarin isolated from Enicostemma axillare. Planta Med 75:12–17

    Article  Google Scholar 

  • Vogeli LR, Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves: implication of a transport function for cadmium-binding peptides. Plant Physiol 92:1086–1093

    Article  Google Scholar 

  • Yannarelli GG, Alvarez AJF, Santa-Cruz DM, Tomaro ML (2007) Glutathione reductase activity and isoforms in leaves and roots of wheat plants subjected to Cd stress. Phytochemistry 68:505–512

    Article  CAS  PubMed  Google Scholar 

  • Yen Duh PD (1993) Antioxidative properties of methanolic extracts from peanut hulls. J Amer Oil Chem Society 70:383–386

    Article  Google Scholar 

  • Zeng X, Ma LQ, Oiu R, Tang Y (2009) Responses of nonprotein thiols to Cd exposure in Cd hyperaccumulator Arabis paniculata Franch. Env Exp Bot 66:242–248

    Article  Google Scholar 

  • Zheng G, Lv HP, Gao S, Wang SR (2010) Effect of Cd on growth and antioxidant response in Glycyrrhiza uralensis seedlings. Plant Soil Environ 56:508–515

    CAS  Google Scholar 

Download references

Acknowledgments

Authors are thankful to the NMITLI, CSIR, New Delhi for the financial assistance. The help by Soil Science department (Drs. Sukhmal Chand, P. Khare, A. Kalra and Mr. Kundan)  in ICP analysis is gratefully acknowledged. BM and NSS thank Academy of Scientific and Innovative Research (AcSIR), New Delhi. Authors are thankful to Director, CSIR-CIMAP for constant encouragement and support.

Conflict of interest

The authors declare they have no conflicts of interest.

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Author notes

  1. Rajender S. Sangwan

    Present address: Center of Innovative and Applied Bioprocessing (CIAB), C-127, Phase 8, Industrial Area, SAS Nagar, Mohali, 160071, Punjab, India

Authors and Affiliations

  1. CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), P.O. CIMAP, Lucknow, 226015, Uttar Pradesh, India

    Bhawana Mishra, Smrati Mishra, Jyoti S. Jadaun, Farzana Sabir & Neelam S. Sangwan

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  1. Bhawana Mishra
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Supplementary Table 1

(DOC 40 kb)

Supplementary Fig. 1A

Effect of Cd supplementation on growth of in vitro shoots of W. somnifera after 30 days of treatment (A control; B 5 μM; C 10 μM; D 20 μM; E 50 μM; F 100 μM; G 150 μM; H 200 μM; I 300 μM). (PDF 140 kb)

Supplementary Fig. 1B

Inhibition of growth and branching in in vitro grown shoots of W. somnifera after 30 days of treatment. a control. b 300 μM (PDF 115 kb)

Supplementary Fig. 2

Effect of Cd supplementation on FW and DW of multiple shoot cultures of W. somnifera after 30 days of treatment. Average values are given. Error bar represents the standard deviation (n = 3). (PDF 26 kb)

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Mishra, B., Sangwan, R.S., Mishra, S. et al. Effect of cadmium stress on inductive enzymatic and nonenzymatic responses of ROS and sugar metabolism in multiple shoot cultures of Ashwagandha (Withania somnifera Dunal). Protoplasma 251, 1031–1045 (2014). https://doi.org/10.1007/s00709-014-0613-4

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