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WsSGTL1 gene from Withania somnifera, modulates glycosylation profile, antioxidant system and confers biotic and salt stress tolerance in transgenic tobacco

Abstract

Glycosylation of sterols, catalysed by sterol glycosyltransferases (SGTs), improves the sterol solubility, chemical stability and compartmentalization, and helps plants to adapt to environmental changes. The SGTs in medicinal plants are of particular interest for their role in the biosynthesis of pharmacologically active substances. WsSGTL1, a SGT isolated from Withania somnifera, was expressed and functionally characterized in transgenic tobacco plants. Transgenic WsSGTL1-Nt lines showed an adaptive mechanism through demonstrating late germination, stunted growth, yellowish-green leaves and enhanced antioxidant system. The reduced chlorophyll content and chlorophyll fluorescence with decreased photosynthetic parameters were observed in WsSGTL1-Nt plants. These changes could be due to the enhanced glycosylation by WsSGTL1, as no modulation in chlorophyll biogenesis-related genes was observed in transgenic lines as compared to wildtype (WT) plants. Enhanced accumulation of main sterols like, campesterol, stigmasterol and sitosterol in glycosylated form was observed in WsSGTL1-Nt plants. Apart from these, other secondary metabolites related to plant’s antioxidant system along with activities of antioxidant enzymes (SOD, CAT; two to fourfold) were enhanced in WsSGTL1-Nt as compared to WT. WsSGTL1-Nt plants showed significant resistance towards Spodoptera litura (biotic stress) with up to 27 % reduced larval weight as well as salt stress (abiotic stress) with improved survival capacity of leaf discs. The present study demonstrates that higher glycosylation of sterols and enhanced antioxidant system caused by expression of WsSGTL1 gene confers specific functions in plants to adapt under different environmental challenges.

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Abbreviations

NTPH:

Nicotiana tabacum cv. Petit Havana

UGT:

Uridine diphosphate glycosyltransferase

WsSGTL1:

Sterol glucosyltransferase gene (Clone1) of Withania somnifera

WsSGTL1-Nt :

WsSGTL1 expressing transgenic plants of Nicotiana tabacum

WT:

Wild type

References

  1. Abdelwahd R, Hakam N, Labhilili M, Udupa SM (2008) Use of an adsorbent and antioxidants to reduce the effects of leached phenolics in in vitro plantlet regeneration of faba bean. Afr J Biotechnol 7:997–1002

  2. Aebi H (1974) Catalases. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 2. Harcourt Brace Javanovich, Germany, pp 673–684

  3. Ahlawat P, Khajuria A, Bhagwat DP, Kalia B (2012) Therapeutic benefits of Withania somnifera: an exhaustive review. Int J Pharm Chem Sci 1:491–496

  4. Akhtar N, Gupta P, Sangwan NS, Sangwan RS, Trivedi PK (2013) Cloning and functional characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Withania somnifera: an important medicinal plant. Protoplasma 250:613–622

  5. Alam N, Hossain M, Mottalib MA, Sulaiman SA, Gan SH, Khalil MI (2012) Methanolic extracts of Withania somnifera leaves, fruits and roots possess antioxidant properties and antibacterial activities. BMC Complement Altern Med 12:175. doi:10.1186/1472-6882-12-175

  6. Alscher RG, Erturk N, Heath LS (2002) Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot 53:1331–1341

  7. Alves PLCA, Magalhaes ACN, Barja PR (2002) The phenomenon of photoinhibition of photosynthesis and its importance in reforestation. Bot Rev 68:193–208

  8. Arnon DI (1949) Copper enzymes in isolated chloroplasts, polyphenoxidase in Beta vulgaris. Plant Physiol 24:1–15

  9. Beyer WF, Fridovich I (1987) Assaying for superoxide dismutase activity: some large consequences of minor changes in condition. Anal Biochem 161:559–566

  10. Bhatia A, Bharti SK, Tewari SK, Sidhu OP, Roy R (2013) Metabolic profiling for studying chemotype variations in Withania somnifera (L.) Dunal fruits using GC–MS and NMR spectroscopy. Phytochemistry 93:105–115

  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 72:248–254

  12. Bush PB, Grunwald C (1972) Sterol changes during germination of Nicotiana tabacum seeds. Plant Physiol 50:69–72

  13. Caputi L, Malnoy M, Goremykin V, Nikiforova S, Martens S (2012) A genome-wide phylogenetic reconstruction of family 1 UDP-glycosyltransferases revealed the expansion of the family during the adaptation of plants to life on land. Plant J 69:1030–1042

  14. Chatterjee S, Srivastava S, Khalid A, Singh N, Sangwan RS, Sidhu OP, Roy R, Khetrapal CL, Tuli R (2010) Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts. Phytochemistry 71:1085–1094

  15. Chaturvedi P, Misra P, Tuli R (2011) Sterol glycosyltransferases--the enzymes that modify sterols. Appl Biochem Biotechnol 165:47-68

  16. Chaturvedi P, Mishra M, Akhtar N, Gupta P, Mishra P, Tuli R (2012) Sterol glycosyltransferases-identification of members of gene family and their role in stress in Withania somnifera. Mol Biol Rep 39:9755–9764

  17. Chaurasiya ND, Sangwan NS, Sabir F, Misra L, Sangwan RS (2012) Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in ashwagandha Withania somnifera L. (Dunal). Plant Cell Rep 31:1889–1897

  18. Chen LX, He H, Qiu F (2011) Natural withanolides: an overview. Nat Prod Rep 28:705–740

  19. Ðinh ST, Gális I, Baldwin IT (2013) UVB radiation and 17-hydroxygeranyllinalool diterpene glycosides provide durable resistance against mirid (Tupiocoris notatus) attack in field-grown Nicotiana attenuata plants. Plant Cell Environ 36:590–606

  20. Dubey S, Misra P, Dwivedi S, Chatterjee S, Bag SK, Mantri S, Asif MH, Rai A, Kumar S, Shri M, Tripathi P, Tripathi RD, Trivedi PK, Chakrabarty D, Tuli R (2010) Transcriptomic and metabolomic shifts in rice roots in response to Cr (VI) stress. BMC Genomics 11:648

  21. Dubouzet JG, Matsuda F, Ishihara A, Miyagawa H, Wakasa K (2013) Production of indole alkaloids by metabolic engineering of the tryptophan pathway in rice. Plant Biotechnol J. doi:10.1111/pbi.12105

  22. Duxbury AC, Yentsch CS (1956) Plankton pigment monographs. J Marine Res 15:91–101

  23. Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research, 2nd edn. Wiley, London, pp 13–175

  24. Grunwald C (1970) Sterol distribution in intracellular organelles isolated from tobacco leaves. Plant Physiol 45:663–666

  25. Grunwald C (1978) Shading influence on the sterol balance of Nicotiana tabacum L. Plant Physiol 61:76–79

  26. Gupta P, Akhtar N, Tewari SK, Sangwan RS, Trivedi PK (2011) Differential expression of farnesyl diphosphate synthase gene from Withania somnifera in different chemotypes and in response to elicitors. Plant Growth Regul 65:93–100

  27. Gupta N, Sharma P, Santosh Kumar RJ, Vishwakarma RK, Khan BM (2012) Functional characterization and differential expression studies of squalene synthase from Withania somnifera. Mol Biol Rep 39:8803–8812

  28. Gupta P, Agarwal AV, Akhtar N, Sangwan RS, Singh SP, Trivedi PK (2013a) Cloning and characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera. Protoplasma 250:285–295

  29. Gupta P, Goel R, Pathak S, Srivastava A, Singh SP, Sangwan RS, Asif MH, Trivedi PK (2013b) De novo assembly, functional annotation and comparative analysis of Withania somnifera leaf and root transcriptomes to identify putative genes involved in the withanolides biosynthesis. PLoS One 8:e62714

  30. Han YJ, Cho KC, Hwang OJ, Choi YS, Shin AY, Hwang I, Kim JI (2012) Overexpression of an Arabidopsis β-glucosidase gene enhances drought resistance with dwarf phenotype in creeping bentgrass. Plant Cell Rep 31:1677–1686

  31. Horsch RB, Fry JE, Hoffmann NL, Eicholtz D, Rogers SG, Fraley RT (1985) A simple method for transferring genes into plants. Science 227:1229–1231

  32. Ibrahim MH, Jaafar HZE (2012) Primary, secondary metabolites, H2O2, malondialdehyde and photosynthetic responses of Orthosiphon stimaneus Benth. to different irradiance levels. Molecules 17:1159–1176

  33. Ihsan-ul-Haq Youn UJ, Chai Xingyun, Park EJ, Kondratyuk TP, Simmons CJ, Borris RP, Mirza B, Pezzuto JM, Chang LC (2013) Biologically active withanolides from Withania coagulans. J Nat Prod 76:22–28

  34. Jadhav DR, Mallikarjuna N, Rathore A, Pokle D (2012) Effect of some flavonoids on survival and development of Helicoverpa armigera (Hübner) and Spodoptera litura (Fab) (Lepidoptera: noctuidae). Asian J Agric Sci 4:298–307

  35. Jones P, Vogt T (2001) Glycosyltransferases in secondary plant metabolism: tranquilizers and stimulant controllers. Planta 213:164–174

  36. Khedgikar V, Kushwaha P, Gautam J, Verma A, Changkija B, Kumar A, Sharma S, Nagar G, Singh D, Trivedi PK, Sangwan NS, Mishra PK, Trivedi R (2013) Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone. Cell Death Dis 4:e778

  37. Kim HS, Kim BG, Sung S, Kim M, Mok H, Chong Y, Ahn JH (2013) Engineering flavonoid glycosyltransferases for enhanced catalytic efficiency and extended sugar-donor selectivity. Planta 238:683–693

  38. Kulkarni SK, Dhir A (2008) Withania somnifera: an Indian ginseng. Prog Neuropsychopharmacol Biol Psychiatry 32:1093–1105

  39. Leiss KA, Maltese F, Choi YH, Verpoorte R, Klinkhamer PGL (2009) Identification of chlorogenic acid as a resistance factor for thrips in Chrysanthemum. Plant Physiol 150:1567–1575

  40. Leyva-González MA, Ibarra-Laclette E, Cruz-Ramírez A, Herrera-Estrella L (2012) Functional and transcriptome analysis reveals an acclimatization strategy for abiotic stress tolerance mediated by Arabidopsis NF-YA family members. PLoS One 7:e48138

  41. Liu WH, Ding B, Ruan XM, Xu HT, Yang J, Liu SM (2007) Analysis of free and conjugated phytosterols in tobacco by an improved method using gas chromatography–flame ionization detection. J Chromatogr A 1163:304–311

  42. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

  43. Maclachlan S, Zalik S (1963) Plastid structure, chlorophyll concentration, and free amino acid composition of a chlorophyll mutant of barley. Can J Bot 41:1053–1062

  44. Madina BR, Sharma LK, Chaturvedi P, Sangwan RS, Tuli R (2007a) Purification and physico-kinetic characterization of 3β-hydroxy specific sterol glucosyltransferase from Withania somnifera (L.) and its stress response. Biochim Biophys Acta 1774:392–402

  45. Madina BR, Sharma LK, Chaturvedi P, Sangwan RS, Tuli R (2007b) Purification and characterization of a novel glucosyltransferase specific to 27β-hydroxy steroidal lactones from Withania somnifera and its role in stress responses. Biochim Biophys Acta 1774:1199–1207

  46. Maizura M, Aminah A, Wan Aida W (2011) Total phenolic content and antioxidant activity of kesum (Polygonum minus), ginger (Zingiber officinale) and turmeric (Curcuma longa) extract. Intl Food Res J 18:529–534

  47. Margaret C, Campbell BC, Godwin ID (2010) Transgenic plants for abiotic stress resistance. In: Kole C, Michler CH, Abbott AG, Hall TC (eds) Transgenic crop plants, 2nd edn. Springer, Heidelberg, pp 67–132

  48. Maxwell K, Johnson G (2000) Chlorophyll fluorescence—a practical guide. J Exp Bot 5:659–668

  49. Mishra LC, Singh BB, Dagenais S (2000) Scientific basis for the therapeutic use of Withania somnifera (Ashwagandha): a review. Alt Med Rev 5:334–346

  50. Mishra MK, Chaturvedi P, Singh R, Singh G, Sharma LK, Pandey V, Kumari N, Misra P (2013a) Overexpression of WsSGTL1 gene of Withania somnifera enhances salt tolerance, heat tolerance and cold acclimation ability in transgenic Arabidopsis plants. PLoS One 8:e63064

  51. Mishra S, Sangwan RS, Bansal S, Sangwan NS (2013b) Efficient genetic transformation of Withania coagulans (Stocks) Dunal mediated by Agrobacterium tumefaciens from leaf explants of in vitro multiple shoot culture. Protoplasma 250:451–458

  52. Misra P, Pandey A, Tewari SK, Nath P, Trivedi PK (2010a) Characterization of isoflavone synthase gene from Psoralea corylifolia: a medicinal plant. Plant Cell Rep 29:747–755

  53. Misra P, Pandey A, Tiwari M, Chandrashekar K, Sidhu OP, Asif MH, Chakrabarty D, Singh PK, Trivedi PK, Nath P, Tuli R (2010b) Modulation of transcriptome and metabolome of tobacco by Arabidopsis transcription factor, AtMYB12, leads to insect resistance. Plant Physiol 152:2258–2268

  54. Mucciarelli M, Scannerini S, Gallino M, Maffei M (2000) Effects of 3,4-dihydroxybenzoic acid on tobacco (Nicotiana tabacum L.) cultured in vitro growth regulation in callus and organ cultures. Plant Biosyst 134:185–192

  55. Murashige T, Skoog FA (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497

  56. Niranjan A, Barthwal J, Lehri A, Singh DP, Govindrajan R, Rawat AKS, Amla DV (2009) Development and validation of an HPLC–UV–MS–MS method for identification and quantification of polyphenols in Artemisia pallens L. Acta Chromatogr 21:105–116

  57. Ozyigit II, Kahraman MV, Ercan O (2007) Relation between explant age, total phenols and regeneration response in tissue cultured cotton (Gossypium hirsutum L.). Afr J Biotechnol 6:3–8

  58. Pandey V, Misra P, Chaturvedi P, Mishra MK, Trivedi PK, Tuli R (2010a) Agrobacterium tumefaciens mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant. Pant Cell Rep 29:133–141

  59. Pandey V, Ranjan S, Deeba F, Pandey AK, Singh R, Shirke PA, Pathre UV (2010b) Desiccation-induced physiological and biochemical changes in resurrection plant, Selaginella bryopteris. J Plant Physiol 167:1351–1359

  60. Pandey A, Misra P, Chandrashekar K, Trivedi PK (2012) Development of AtMYB12-expressing transgenic tobacco callus culture for production of rutin with biopesticidal potential. Plant Cell Rep 31:1867–1876

  61. Pandey A, Misra P, Khan MP, Swarnkar G, Tewari MC, Bhambhani S, Trivedi R, Chattopadhyay N, Trivedi PK (2013) Co-expression of Arabidopsis transcription factor, AtMYB12, and soybean isoflavone synthase, GmIFS1, genes in tobacco leads to enhanced biosynthesis of isoflavones and flavonols resulting in osteoprotective activity. Plant Biotechnol J. doi:10.1111/pbi.12118

  62. Pawlowski K, Kunze R, Vries SD, Bisseling T (1994) Isolation of total poly (A) and polysomal RNA from plant tissue. In: Gelvin SB, Schilperoort RA (ed) Plant Mol Biol Manual, vol D5. Kluwer Academic Publishers, Belgium, pp 213–243

  63. Pütter J (1974) Peroxidases. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 2. Harcourt Brace Javanovich, Germany, pp 685–690

  64. Qiu J, Gao F, Shen G, Li C, Han X, Zhao Q, Zhao D, Hua X, Pang Y (2013) Metabolic engineering of the phenylpropanoid pathway enhances the antioxidant capacity of Saussurea involucrata. PLoS One 8:e70665

  65. Razdan S, Bhat WW, Rana S, Dhar N, Lattoo SK, Dhar RS, Vishwakarma RA (2013) Molecular characterization and promoter analysis of squalene epoxidase gene from Withania somnifera (L.) Dunal. Mol Biol Rep 40:905–916

  66. Sayama T, Ono E, Takagi K, Takada Y, Horikawa M, Nakamoto Y, Hirose A, Sasama H, Ohashi M, Hasegawa H, Terakawa T, Kikuchi A, Kato S, Tatsuzaki N, Tsukamoto C, Ishimoto M (2012) The Sg-1 glycosyltransferase locus regulates structural diversity of triterpenoid saponins of soybean. Plant Cell 24:2123–2138

  67. Sharma LK, Madina BR, Chaturvedi P, Sangwan RS, Tuli R (2007) Molecular cloning and characterization of one member of 3b-hydroxy sterol glucosyltransferase gene family in Withania somnifera. Arch Biochim Biophys 460:48–55

  68. Sharma V, Sharma S, Paliwal R, Pracheta (2011) Withania somnifera: a rejuvenating, ayurvedic medicinal herb for the treatment of various human ailments. Int J Pharm Tech Res 3:187–192

  69. Shimamura M (2012) Immunological functions of steryl glycosides. Arch Immunol Ther Exp 60:351–359

  70. Siddiqui Z (2013) Effects of double stress on antioxidant enzyme activity in Vigna radiata (L.) Wilczek. Acta Bot Croat 72:145–156

  71. Uddin Q, Samiulla L, Singh VK, Jamil SS (2012) Phytochemical and pharmacological profile of Withania somnifera Dunal: a review. J Appl Pharm Sci 2:170–175

  72. Veljanovski V, Constabel CP (2013) Molecular cloning and biochemical characterization of two UDP-glycosyltransferases from poplar. Phytochemistry 91:148–157

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Acknowledgments

The Director, Council of Scientific and Industrial Research, National Botanical Research Institute, is gratefully acknowledged by authors for the provided best facilities and moral support. PM is thankful to the Department of Biotechnology (Project No. GAP 231225), Govt. of India, for providing financial support to carry out the research work and CSIR-NMITLI project for identification of WsSGTL1 gene. VP is thankful to CSIR for the award of Senior Research Fellowship.

Author information

Correspondence to Pratibha Misra.

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Supplementary Fig. S1 Stomatal conductance, gs (a) and chlorophyll quantification (b) from leaves of 3-month-old WsSGTL1-Nt (L1, L2, L3) and WT plants. Data are expressed as mean ± SD of three independent experiments. Different letters indicate significantly different values in a particular tissue (DMRT, P ≤ 0.05) and ANOVA significant at P ≤ 0.01 (TIFF 124 kb)

Supplementary Fig. S2 Expression analysis of some nuclear encoded genes related to chlorophyll biogenesis in WsSGTL1-Nt and WT. No significant difference was observed in the expression of these genes, which suggested that stress adaptation is not associated with chlorophyll biogenesis while in WT were relatively lower, i.e. 46.7, 3.9 μg g−1 DW and 11 mg g−1 DW, respectively. Three independent experiments were performed (TIFF 253 kb)

Supplementary Fig. S3 Average seed (100 seeds) weight (a) was calculated for seeds of WsSGTL1-Nt and WT, showing no significant difference in yield. Also, no significant difference was observed during cotyledon emergence (b) of WsSGTL1-Nt and WT seeds. Data are expressed as mean ± SD of three independent experiments. Different letters indicate significantly different values in a particular tissue (DMRT, P ≤ 0.05) and ANOVA significant at P ≤ 0.01 (TIFF 74 kb)

Supplementary Fig. S4 HPLC profiling of seed extract of WsSGTL1-Nt (L1, L2 and L3 lines) and wild type plants illustrating the more glycosylated sterols in WsSGTL1-Nt. Standards of 1: campesterol; 2: stigmasterol; 3: sitosterol (a) before hydrolysis (b) after hydrolysis (c) (TIFF 393 kb)

Supplementary Fig. S5 HPLC profiling of leaf extract of WsSGTL1-Nt (L1, L2 and L3 lines) and WT showing enhanced accumulation of phenolics in transgenic lines as compared to wild type. a Standards (1: Gallic Acid; 2: Protocetechric Acid; 3:Chlorogenic Acid; 4: Coffeic Acid; 5: Rutin; 6: Ferulic Acid; 7: Quercetin; 8: Kaempherol). be L1, L2 and L3 lines of WsSGTL1-Nt (b, c, d) and wild type plants (e) (TIFF 226 kb)

SupplementaryFig. S6 Quantitative estimation of some phenolics which may serve as substrates (resulted in glycosylated products). HPLC quantification reveals no significant change in accumulation of gallic acid (GA) metabolites in WsSGTL1-Nt and WT, while amount of other secondary metabolites like, kempherol (K), ferulic acid (FA) and caffeic acid (CA) decreased significantly in correspondence to their enhanced glycosylated form in WsSGTL1-Nt as compared to WT plants (TIFF 108 kb)

Supplementary Fig. S7 Quantification of chlorophyll contents; chlorophyll-A, chlorophyll-B, carotenoid in the leaf-disc of WsSGTL1-Nt and WT were carried out during 0 to 6 day of 200 mM NaCl treatment, showing enhanced salt tolerance capacity of transgenic lines as lesser chlorosis in WsSGTL1-Nt. Data are expressed as mean ± SD of three independent experiments. Different letters indicate significantly different values in a particular tissue (DMRT, P ≤ 0.05) and ANOVA significant at P ≤ 0.01 (TIFF 143 kb)

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Pandey, V., Niranjan, A., Atri, N. et al. WsSGTL1 gene from Withania somnifera, modulates glycosylation profile, antioxidant system and confers biotic and salt stress tolerance in transgenic tobacco. Planta 239, 1217–1231 (2014). https://doi.org/10.1007/s00425-014-2046-x

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Keywords

  • Adaptation
  • Antioxidant
  • Glycosylation
  • Environmental stress
  • Secondary metabolites