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Transformation of Lettuce with rol ABC Genes: Extracts Show Enhanced Antioxidant, Analgesic, Anti-Inflammatory, Antidepressant, and Anticoagulant Activities in Rats

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Abstract

Lettuce is an edible crop that is well known for dietary and antioxidant benefits. The present study was conducted to investigate the effects of rol ABC genes on antioxidant and medicinal potential of lettuce by Agrobacterium-mediated transformation. Transgene integration and expression was confirmed through PCR and real-time RT-PCR, respectively. The transformed plants showed 91–102 % increase in total phenolic contents and 53–65 % increase in total flavonoid contents compared to untransformed plants. Total antioxidant capacity and total reducing power increased up to 112 and 133 % in transformed plants, respectively. Results of DPPH assay showed maximum 51 % increase, and lipid peroxidation assay exhibited 20 % increase in antioxidant activity of transformed plants compared to controls. Different in vivo assays were carried out in rats. The transgenic plants showed up to 80 % inhibition in both hot plate analgesic assay and carrageenan-induced hind paw edema test, while untransformed plants showed only 45 % inhibition. Antidepressant and anticoagulant potential of transformed plants was also significantly enhanced compared to untransformed plants. Taken together, the present work highlights the use of rol genes to enhance the secondary metabolite production in lettuce and improve its analgesic, anti-inflammatory, antidepressant, and anticoagulatory properties.

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References

  1. Lucier, G., & Jerardo, A. (2005). Vegetables and melons situation and outlook yearbook. Electronic Outlook Report from the Economic Research Service USDA, 1–42.

  2. Ismail, H., & Mirza, B. (2015). Evaluation of analgesic, anti-inflammatory, anti-depressant and anti-coagulant properties of Lactuca sativa (CV. Grand Rapids) plant tissues and cell suspension in rats. BMC Complementary and Alternative Medicine, 15(1), 199.

    Article  Google Scholar 

  3. Yakoot, M., Helmy, S., & Fawal, K. (2011). Pilot study of the efficacy and safety of lettuce seed oil in patients with sleep disorders. International Journal of General Medicine, 4, 451–456.

    Article  Google Scholar 

  4. Seeram, N. P., Aviram, M., Zhang, Y., Henning, S. M., Feng, L., Dreher, M., & Heber, D. (2008). Comparison of antioxidant potency of commonly consumed polyphenol-rich beverages in the United States. Journal of Agricultural and Food Chemistry, 56(4), 1415–1422.

    Article  CAS  Google Scholar 

  5. Harsha, S. N., & Anilakumar, K. R. (2013). Anxiolytic property of hydro-alcohol extract of Lactuca sativa and its effect on behavioral activities of mice. Journal of Biomedical Research, 27(1), 37–42.

    Article  CAS  Google Scholar 

  6. Jaiswal, J. V., Wadegaonkar, P. A., & Hajare, S. W. (2012). The bioflavonoid galangin suppresses the growth of Ehrlich ascites carcinoma in swiss albino mice: a molecular insight. Applied Biochemistry and Biotechnology, 167, 1325–1339.

    Article  CAS  Google Scholar 

  7. Caldwell, C. R. (2003). Alkylperoxyl radical scavenging activity of red leaf lettuce (Lactuca sativa L.) phenolics. Journal of Agricultural and Food Chemistry, 51, 4589–4595.

    Article  CAS  Google Scholar 

  8. Liu, X., Ardo, S., Bunning, M., Parry, J., Zhou, K., Stushnoff, C., et al. (2007). Total phenolic content and DPPH radical scavenging activity of lettuce (Lactuca sativa L.) grown in Colorado. LWT-Food Science and Technology, 40, 552–557.

    Article  CAS  Google Scholar 

  9. DuPont, M. S., Mondin, Z., Williamson, G., & Price, K. R. (2000). Effect of variety, processing and storage on the flavonoid glycoside content and composition of lettuce endive. Journal of Agricultural and Food Chemistry, 48, 3957–3964.

    Article  CAS  Google Scholar 

  10. Ferreres, F., Gil, I., Castan, M., & Toma, F. A. (1997). Phenolic metabolites in red pigmented lettuce (Lactuca sativa). Changes with minimal processing and cold storage. Journal of Agricultural and Food Chemistry, 45(97), 4249–4254.

    Article  Google Scholar 

  11. Deng, X., Zhou, Y., & Chang, J. (2007). Establishment of genetic transformation system and transgenic studies in lettuce (Lactuca sativa var. capatata). Acta. Bot. Yunna, 29(1), 29(1), 98–102.

    CAS  Google Scholar 

  12. Song, L., Zhao, D., Wu, Y., & Li, Y. (2008). Transient expression of chicken alpha interferon gene in lettuce. Journal of Zhejiang University. Science. B, 9(5), 351–355.

    CAS  Google Scholar 

  13. Huy, N.-X., Yang, M.-S., & Kim, T.-G. (2011). Expression of a cholera toxin B subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (Lactuca sativa L.). Molecular Biotechnology, 48(3), 201–209.

    Article  CAS  Google Scholar 

  14. Meng, Z., Zhang, R., Liu, D., Yan, X., Geng, Q., & Guo, S. (2005). Expression of human lactoferrin, thymosin fusion gene in lettuce. National symposium abstract. Crop Biotechnol. Mutagen. Technol., 25.

  15. Tian, X. (2007). A study of transformation of nattokinase into lettuce. M.D. Thesis Lanzhou University.

  16. Li, X., Li, X., Zhang, J., Niu, J., Guo, R., Yang, H., & Liu, L. (2006). Expression and inheritance of lysine rich potein gene in lettuce (Lactuca sativa L.). Chinese Journal of Applied and Environmental Biology, 12(4), 472–475.

    CAS  Google Scholar 

  17. Kim, T.-G., Kim, M.-Y., Kim, B.-G., Kang, T.-J., Kim, Y.-S., Jang, Y.-S., et al. (2007). Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic lettuce (Lactuca sativa). Protein Expression and Purification, 51(1), 22–27.

    Article  CAS  Google Scholar 

  18. Wang, W., Guo, X., & Tang, K. (2011). Transformation of GDP-mannose pyrophosphorylase gene from Arabidopsis thaliana L. into Lactuca sativa L. Journal of Shanghai Jiaotong University (Agricultural Science), 29(2), 43–49.

    Google Scholar 

  19. Waheed, M. T., Ismail, H., Gottschamel, J., Mirza, B., & Lossl, A. G. (2015). Plastids: the green frontiers for vaccine production. Frontiers in Plant Science, 6, 1–11.

    Article  Google Scholar 

  20. Lossl, A. G., & Waheed, M. T. (2011). Chloroplast-derived vaccines against human diseases: achievements, challenges and scopes. Plant Biotechnology Journal, 9, 527–539.

    Article  Google Scholar 

  21. Sobolev, A. P., Segre, A. L., Giannino, D., Mariotti, D., Nicolodi, C., Brosio, E., & Amato, M. E. (2007). Strong increase of foliar inulin occurs in transgenic lettuce plants (Lactuca sativa L.) overexpressing the asparagine synthetase A gene from Escherichia coli. Journal of Agricultural and Food Chemistry, 55(26), 10827–10831.

    Article  CAS  Google Scholar 

  22. Han, E., Lee, J., Lee, J., Chung, I., & Lee, Y. (2011). Transgenic lettuce expressing chalcone isomerase gene of chinese cabbage increased levels of flavonoids and polyphenols. Korean Journal of Horticultural Science & Technology, 29(August), 467–473.

    CAS  Google Scholar 

  23. Landi, M., Fambrini, M., Basile, A., Salvini, M., Guidi, L., & Pugliesi, C. (2015). Overexpression of L-galactono-1,4-lactone dehydrogenase (L-GalLDH) gene correlates with increased ascorbate concentration and reduced browning in leaves of Lactuca sativa L. after cutting. Plant Cell, Tissue and Organ Culture (PCTOC), 123(1), 109–120.

    Article  CAS  Google Scholar 

  24. Jimenez-Garcia, S. N., Torres-Pacheco, I., & Andres Cruz-Hernandez, A. F.-P. (2013). Current approaches for enhanced expression of secondary metabolites as bioactive compounds in plants for agronomic and human health purposes—a review. Polish Journal of Food and Nutrition Sciences, 63(2), 67–78.

    Article  CAS  Google Scholar 

  25. Bulgakov, V. P. (2008). Functions of rol genes in plant secondary metabolism. Biotechnology Advances, 26(4), 318–324.

    Article  CAS  Google Scholar 

  26. Dilshad, E., Zafar, S., Ismail, H., Waheed, M. T., Cusido, R. M., Palazon, J., & Mirza, B. (2016). Effect of rol genes on polyphenols biosynthesis in Artemisia annua and their effect on antioxidant and cytotoxic potential of the plant. Applied Biochemistry and Biotechnology, 1–13.

  27. Ismail, H., Dilshad, E., Waheed, M. T., Sajid, M., Kayani, W. K., & Mirza, B. (2016). Transformation of Lactuca sativa L. with rol C gene results in increased antioxidant potential and enhanced analgesic, anti-inflammatory and antidepressant activities in vivo. 3 Biotech (Accepted).

  28. Nijveldt, R. J., Van Nood, E., Van Hoorn, D. E. C., Boelens, P. G., Van Norren, K., & Van Leeuwen, P. A. M. (2001). Flavonoids: a review of probable mechanisms of action and potential applications. American Journal of Clinical Nutrition, 74(4), 418–425.

    CAS  Google Scholar 

  29. Erlejman, A. G., Fraga, C. G., & Oteiza, P. I. (2006). Procyanidins protect CaCo-2 cells from bile acid- and oxidant-induced damage. Free Radical Biology and Medicine, 41, 1247–1256.

    Article  CAS  Google Scholar 

  30. Hodgson, J. M., & Croft, K. D. (2006). Revista de fitoterapia: Editorial. Journal of the Science of Food and Agriculture, 86, 2492–2498.

    Article  CAS  Google Scholar 

  31. Montefusco-Pereira, C. V., de Carvalho, M. J., de Araújo Boleti, A. P., Teixeira, L. S., Matos, H. R., & Lima, E. S. (2013). Antioxidant, anti-inflammatory, and hypoglycemic effects of the leaf extract from Passiflora nitida Kunth. Applied Biochemistry and Biotechnology, 170(6), 1367–1378.

  32. Spena, A., Schmülling, T., Koncz, C., & Schell, J. S. (1987). Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants. The EMBO Journal, 6(13), 3891–3899.

    CAS  Google Scholar 

  33. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15, 473–497.

    Article  CAS  Google Scholar 

  34. Torres, A. C., Cantliffe, D. J., Laughner, B., Bieniek, M., Nagata, R., Ashraf, M., & Ferl, R. J. (1993). Stable transformation of lettuce cultivar South Bay from cotyledon explants. Plant Cell, Tissue and Organ Culture, 34, 279–285.

    Article  CAS  Google Scholar 

  35. Clarke, J. D. (2009). Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation. Cold Spring Harbor Protocols, 4(3), pdb.prot5177–pdb.prot5177.

  36. Kiani, B. H., Safdar, N., Mannan, A., & Mirza, B. (2012). Comparative artemisinin analysis in Artemisia dubia transformed with two different Agrobacteria harbouring rol ABC genes. Plant Omics Journal, 5(4), 386–391.

    CAS  Google Scholar 

  37. Dilshad, E., Ismail, H., Kayani, W. K., & Mirza, B. (2016). Optimization of conditions for genetic transformation and in vitro propagation of Artemisia carvifolia Buch. Current Synthetic and Systems Biology, 4(1), 1–5.

    Google Scholar 

  38. Wroblewski, T., Piskurewicz, U., Tomczak, A., Ochoa, O., & Michelmore, R. W. (2007). Silencing of the major family of NBS-LRR-encoding genes in lettuce results in the loss of multiple resistance specificities. Plant Journal, 51(5), 803–818.

    Article  CAS  Google Scholar 

  39. Moein, M. R., Moein, S., & Ahmadizadeh, S. (2008). Radical scavenging and reducing power of Salvia mirzayanii subfractions. Molecules, 13, 2804–2813.

    Article  CAS  Google Scholar 

  40. Phatak, R. S., & Hendre, A. S. (2014). Total antioxidant capacity (TAC) of fresh leaves of Kalanchoe pinnata. Journal of Pharmacognosy and Phytochemistry, 2(5), 32–35.

    Google Scholar 

  41. Gülen, H., Çetinkaya, C., Kadıoğlu, M., Kesici, M., Cansev, A., & Eriş, A. (2008). Peroxidase activity and lipid peroxidation in strawberry (Fragaria X ananassa) plants under low temperature. Journal of Biological & Environmental Science, 2(6), 95–100.

    Google Scholar 

  42. Ismail, H., Mirza, B., Haq, I., Shabbir, M., Akhter, Z., & Basharat, A. (2015). Synthesis, characterization, and pharmacological evaluation of selected aromatic amines. Journal of Chemistry, 2015, 1–9.

    Article  Google Scholar 

  43. Shabbir, M., Akhter, Z., Ahmad, I., Ahmed, S., Ismail, H., Mirza, B., et al. (2016). Synthesis, characterization, biological and electrochemical evaluation of novel ether based ON donor bidentate Schiff bases. Journal of Molecular Structure, 1116, 84–92.

    Article  CAS  Google Scholar 

  44. Eddy, N. B., & Leimbach, D. (1952). Synthetic analgesic. II. Dithienylbutenyl- and dithienylbutylamines. Journal of Pharmacology and Experimental Therapeutics, 107, 385–393.

    Google Scholar 

  45. Winter, C. A., Risley, E. A., & Nuss, G. W. (1962). Carrageenin-induced edema in hind paw of the rat as an assay for antiinflammatory drugs. Experimental Biology and Medicine, 111(3), 544–547.

    Article  CAS  Google Scholar 

  46. Slattery, D. A., & Cryan, J. F. (2012). Using the rat forced swim test to assess antidepressant-like activity in rodents. Nature Protocols, 7(6), 1009–1014.

    Article  CAS  Google Scholar 

  47. Hosoki, T., & Asahira, T. (1980). In vitro propagation of bromeliads in liquid culture. Horticulture Science, 15, 603–604.

    Google Scholar 

  48. Bustin, S. a. (2002). Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. Journal of Molecular Endocrinology, 29(1), 23–39.

    Article  CAS  Google Scholar 

  49. Curtis, I. S., He, C., Power, J. B., Mariotti, D., Laat, A. D., & Davey, M. R. (1996). The effects of Agrobacterium rhizogenes rolAB genes in lettuce. Plant Science, 115, 123–135.

    Article  CAS  Google Scholar 

  50. Casanova, E., Trillas, M. I., Moysset, L., & Vainstein, A. (2005). Influence of rol genes in floriculture. Biotechnology Advances, 23, 3–39.

    Article  CAS  Google Scholar 

  51. Gharibi, S., Tabatabaei, B. E. S., Saeidi, G., & Goli, S. A. H. (2015). Effect of drought stress on total phenolic, lipid peroxidation, and antioxidant activity of Achillea species. Applied Biochemistry and Biotechnology, 178(4), 796–809.

    Article  Google Scholar 

  52. Sun, J., Chu, Y. F., Wu, X. Z., & Liu, R. H. (2002). Antioxidant and antiproliferative activities of vegetables. Journal of Agricultural and Food Chemistry, 50, 6910–6916.

    Article  Google Scholar 

  53. Pandey, K. B., & Rizvi, S. I. (2009). Plant polyphenols as dietary antioxidants in human health and disease. Oxidative Medicine and Cellular Longevity, 2(5), 270–278.

    Article  Google Scholar 

  54. Namiesnik, J., Vearasilp, K., Nemirovski, A., Leontowicz, H., Leontowicz, M., Pasko, P., et al. (2014). In vitro studies on the relationship between the antioxidant activities of some berry extracts and their binding properties to serum albumin. Applied Biochemistry and Biotechnology, 172(6), 2849–2865.

    Article  CAS  Google Scholar 

  55. Barku, V. Y., Opoku-Boahen, Y., Owusu-Ansah, E., & Mensah, E. F. (2013). Antioxidant activity and the estimation of total phenolic and flavonoid contents of the root extract of Amaranthus spinosus. Asian Journal of Plant Science and Research, 3(1), 69–74.

    CAS  Google Scholar 

  56. Jha, D. K., Panda, L., Ramaiah, S., & Anbarasu, A. (2014). Evaluation and comparison of radical scavenging properties of solvent extracts from Justicia adhatoda leaf using DPPH assay. Applied Biochemistry and Biotechnology, 174(7), 2413–2425.

    Article  CAS  Google Scholar 

  57. Vinson, J. a., Su, X., Zubik, L., & Bose, P. (2001). Phenol antioxidant quantity and quality in foods: fruits. Journal of Agricultural and Food Chemistry, 49(11), 5315–5321.

    Article  CAS  Google Scholar 

  58. Shabbir, M., Akhter, Z., Ahmad, I., Ahmed, S., Ismail, H., Mirza, B., et al. (2015). Synthesis, biological and electrochemical evaluation of novel nitroaromatics as potential anticancerous drugs. Bioelectrochemistry, 104, 85–92.

    Article  CAS  Google Scholar 

  59. Jun, T., Liancai, Z., & Bochu, W. (2007). Effects of quercetin on DNA damage induced by copper ion. International Journal of Pharmacology, 3(1), 19–26.

    Article  Google Scholar 

  60. Vongtau, H. O., Abbah, J., Mosugu, O., Chindo, B. a., Ngazal, I. E., Salawu, a. O., & Gamaniel, K. S. (2004). Antinociceptive profile of the methanolic extract of Neorautanenia mitis root in rats and mice. Journal of Ethnopharmacology, 92, 317–324.

    Article  CAS  Google Scholar 

  61. Richardson, J. D., Aanonsen, L., & Hargreaves, K. M. (1998). Antihyperalgesic effects of spinal cannabinoids. European Journal of Pharmacology, 345, 145–153.

    Article  CAS  Google Scholar 

  62. Suh, N., Honda, T., Finlay, H. J., Barchowsky, A., Williams, C., Benoit, N. E., et al. (1998). Novel triterpenoids suppress inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in mouse macrophages. Cancer Research, 58, 717–723.

    CAS  Google Scholar 

  63. Astuti, K. W., Putu, L., & Larasanty, F. (2013). Combined effects of noni fruit extract (Morinda citrifolia L.) and warfarin on bleeding and coagulation time of mice. International Journal of Pharmacy Teaching & Practices, 4(4), 863–866.

    Google Scholar 

  64. Raaof, A., Al-naqqash, Z. A., Jawad, A. M., & Muhsan, S. M. (2013). Evaluation of the activity of crude alkaloids extracts of Zingiber officinale roscoe., Thymus vulgaris L. and Acacia arabica L. as coagulant agent in lab mice. Biomedicine and Biotechnology, 1(2), 11–16.

    CAS  Google Scholar 

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Acknowledgments

HI acknowledges the Higher Education Commission (HEC), Islamabad, Pakistan, for provision of the scholarship during the study.

Authors’ Contributions

All authors contributed to the work. BM conceived the study design and supervised the study. MTW contributed in the study design and transformation. HI and ED conducted transformation and assays. All authors drafted the manuscript and approved the final version.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Gujrat, Gujrat, 50700, Pakistan

    Hammad Ismail

  2. Department of Bioinformatics and Biosciences, Capital University of Science and Technology, Islamabad, Pakistan

    Erum Dilshad

  3. Department of Biochemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan

    Mohammad Tahir Waheed & Bushra Mirza

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  1. Hammad Ismail
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  2. Erum Dilshad
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  4. Bushra Mirza
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Correspondence to Bushra Mirza.

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The study design was approved by the Institutional Animal Ethics Committee, and all provisions were carried to minimize animal sufferings.

Competing Interests

The scholarship was provided to HI by the Higher Education Commission (HEC), Pakistan, to conduct the study. The funding body has no role in study design, collection, analysis, interpretation of data, and writing of the manuscript and in the decision to submit the manuscript for publication.

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Ismail, H., Dilshad, E., Waheed, M.T. et al. Transformation of Lettuce with rol ABC Genes: Extracts Show Enhanced Antioxidant, Analgesic, Anti-Inflammatory, Antidepressant, and Anticoagulant Activities in Rats. Appl Biochem Biotechnol 181, 1179–1198 (2017). https://doi.org/10.1007/s12010-016-2277-3

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