Advertisement

Physiological and morphological analyses of Thymus vulgaris L. in vitro cultures under polyethylene glycol (PEG)-induced osmotic stress

  • Roya Razavizadeh
  • Farnaz Farahzadianpoor
  • Fatemeh Adabavazeh
  • Setsuko KomatsuEmail author
Plant Tissue Culture
  • 136 Downloads

Abstract

Morphological and physiological parameters of polyethylene glycol (PEG)-induced drought-stressed Thymus vulgaris L. (thyme) seedlings were examined. To start in vitro selection for drought stress tolerance and to provide a more efficient method for the production of pharmaceutical substances, callus induction and production of essential oils were analyzed. Thyme seeds were germinated on medium containing 0, 2, 4, 6, and 8% (w/v) PEG; and, for callus induction, stem explants were cultured on medium supplemented with 1 mg L−1 2,4-dichlorophenoxyacetic acid with different concentrations of PEG. Four weeks after PEG-induced water stress, a significant decrease in seedling growth, chlorophyll and carotenoid contents, and callus weight and induction was observed. However, no significant difference was found in the germination rate of seeds. Organic osmolytes, e.g., proline and carbohydrates, were influenced by PEG-induced osmotic stress. Reducing sugars progressively increased with rising PEG concentrations, while proline levels largely decreased under severe water stress. Moreover, PEG decreased the anthocyanin content but raised reactive oxygen species (ROS), lipid peroxidation, proteins, and phenolic compound levels. Additionally, PEG induced a substantial change in the activities of antioxidant enzymes. Positive relationships between ascorbate peroxidase activity and formation of ROS in PEG-treated thyme seedlings were detected. Furthermore, PEG changed the essential oil composition in seedlings and calluses by significantly increasing γ-terpinene, p-cymene, and geraniol but decreasing thymol and carvacrol. Thus, mild stress levels increased phenolics and oil components in thyme, despite the extent of oxidative injury and growth reduction, which could enhance secondary metabolite production in vitro.

Keywords

Antioxidant enzymes Essential oils Phenolic compounds Polyethylene glycol Callus culture 

Notes

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Aebi H (1974) Catalases. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Academic Press, New York, NY, pp 673–684CrossRefGoogle Scholar
  2. Amenu D (2014) Antimicrobial activity of medicinal plant extracts and their synergistic effect on some selected pathogens. Am J Ethnomed 1:018–029Google Scholar
  3. Asada K, Takahashi M (1987) Production and scavenging of active oxygen in photosynthesis. In: Kyle DJ, Osmond CB, Arntzen CJ (eds) Photoinhibition. Elsevier, Amsterdam, The Netherlands, pp 227–287Google Scholar
  4. Attipali RR, Kolluru VC, Munusamy V (2004) Drought induced responses of photosynthesis and antioxidant metabolism in higher plants. J Plant Physiol 161:1189–1202CrossRefGoogle Scholar
  5. Ayala-Astorga GI, Alcaraz-Meléndez L (2010) Salinity effects on protein content, lipid peroxidation, pigments, and proline in Paulownia imperialis (Siebold and Zuccarini) and Paulownia fortunei (Seemann and Hemsley) grown in vitro. Electron J Biotechnol 13:1–15CrossRefGoogle Scholar
  6. Bates LS, Waldern RP, Teare ID (1973) Rapid determination of free proline for water stress studies. J Plant Soil 39:205–207CrossRefGoogle Scholar
  7. Baxter A, Mittler R, Suzuki N (2014) ROS as key players in plant stress signaling. J Exp Bot 65:1229–1240CrossRefGoogle Scholar
  8. Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assay applicable to acrylamide gels. Ann Biochem 44:276–287CrossRefGoogle Scholar
  9. Benzie FF, Strain JJ (1996) The ferric reducing ability of plasma as leisure of antioxidant power. The FRAP assay. Ann Biochem 239:70–76CrossRefGoogle Scholar
  10. Bettaieb I, Zakhama N, Aidi Wannes W, Marzouk B (2009) Water deficit effects on Salvia officinalis fatty acids and essential oils composition. Sci Hortic 120:271–275CrossRefGoogle Scholar
  11. Bian S, Jiang Y (2009) Reactive oxygen species, antioxidant enzyme activities and gene expression patterns in leaves and roots of Kentucky bluegrass in response to drought stress and recovery. Sci Hortic 120:264–270CrossRefGoogle Scholar
  12. Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress. Ann Bot 91:179–194CrossRefGoogle Scholar
  13. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Annu Rev Biochem 72:248–254CrossRefGoogle Scholar
  14. Chazen O, Hartung W, Neumann PM (1995) The different effects of PEG 6000 and NaCl on leaf development are associated with differential inhibition of root water transport. Plant Cell Environ 18:727–735CrossRefGoogle Scholar
  15. Chen SY, Xiao S, Zhang MX, Chen T, Wang HC, An LZ (2005) Antisense and RNAi expression for a chloroplastic superoxide dismutase gene in transgenic plants. Bot Bull Acad Sin 46:175–182Google Scholar
  16. Close TJ (1996) Dehydrins emergence of a biochemical role of a family of plant dehydration proteins. Physiol Plant 97:795–803CrossRefGoogle Scholar
  17. Creissen G, Firmin J, Fryer M, Kular B, Leyland N, Reynolds H, Pastori G, Wellburn F, Baker N, Wellburn A, Mullineaux P (1999) Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress. Plant Cell 11:1277–1292CrossRefGoogle Scholar
  18. Davies NW (1990) Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methylsilicon and carbowax 20M phases. J Chromatogr 503:1–24CrossRefGoogle Scholar
  19. Davis PH (1982) Flora of Turkey and the east Aegean islands. By Davis PH, assisted by Edmondson JR (ed) University Press, Edinburgh, UK 7, pp 320–354Google Scholar
  20. Ebrahimzadeh MA, Nabavi SM, Nabavi SF, Eslami SH (2010) Antioxidant and free radical scavenging activities of culinary-medicinal mushrooms, golden chanterelle Cantharellus cibarius and Angel’s wings Pleurotus porrigens. Int J Med Mushrooms 12:265–272CrossRefGoogle Scholar
  21. Efferth T (2019) Biotechnology applications of plant callus cultures. Engineering 5:50–59CrossRefGoogle Scholar
  22. Foyer CH, Descourvieres P, Kunert KJ (1994) Photo oxidative stress in plants. Plant Physiol 92:696–717CrossRefGoogle Scholar
  23. Gholami M, Rahemi M, Kholdebarin B, Rastegar S (2012) Biochemical responses in leaves of four fig cultivars subjected to water stress and recovery. Sci Hortic 148:109–117CrossRefGoogle Scholar
  24. Giannotolitis CN, Ries SK (1997) Superoxide dismutase: II. Purification and quantitative relationship with water-soluble protein in seedling. Plant Physiol 59:315–318CrossRefGoogle Scholar
  25. Gray C, Collins J, Gebicki JM (1999) Hydroperoxide assay with the ferric xylenol orange complex. Anal Biochem 273:149–155CrossRefGoogle Scholar
  26. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198CrossRefGoogle Scholar
  27. Hodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid reactive substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604–611CrossRefGoogle Scholar
  28. Huang R, Xia R, Lu Y, Hu L, Xu Y (2008) Effect of preharvest salicylic acid spray treatment on post-harvest antioxidant in the pulp and peel of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck). J Sci Food Agric 88:229–236CrossRefGoogle Scholar
  29. Hund A, Ruta N, Liedgens M (2009) Rooting depth and water use efficiency of tropical maize inbred lines, differing in drought tolerance. Plant Soil 318:311–325CrossRefGoogle Scholar
  30. Janarthanam B, Gopalakrishnan M, Sekar T (2010) Secondary metabolite production in callus cultures of stevia rebaudiana Bertoni. Bangladesh J Sci Ind Res 45:243–248CrossRefGoogle Scholar
  31. Joseph B, Jini D (2011) Development of salt stress-tolerant plants by gene manipulation of antioxidant enzymes. Asian J Agric Res 5:17–27Google Scholar
  32. Karuppusamy S (2009) A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures. J Med Plant Res 3:1222–1239Google Scholar
  33. Lei YB, Yin CY, Li CY (2006) Differences in some morphological, physiological, and biochemical responses to drought stress in two contrasting populations of Populus przewalskii. Physiol Plant 127:182–191CrossRefGoogle Scholar
  34. Li XM, Tian SL, Pang ZC (2009) Extraction of Cuminum cyminum essential oil by combination technology of organic solvent with low boiling point and system distillation. Food Chem 115:1114–1119CrossRefGoogle Scholar
  35. Lia HB, Wonga CC, Chenga KW, Chena F (2008) Antioxidant properties in vitro and total phenolic contents in methanol extracts from medicinal plants. LWT Food Sci Technol 41:385–390CrossRefGoogle Scholar
  36. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic bio membranes. Methods Enzymol 148:350–382CrossRefGoogle Scholar
  37. Lopez-Huertas E, Charlton WL, Johnson B, Graham IA, Baker A (2000) Stress induces peroxisome biogenesis genes. EMBO J 19:6770–6777CrossRefGoogle Scholar
  38. Loreto F, Schnitzler JP (2010) Abiotic stresses and induced BVOCs. Trends Plant Sci 15:154–166CrossRefGoogle Scholar
  39. Lutts S, Almansouri M, Kinet J (2004) Salinity and water stress have contrasting effect on the relationship between growth and cell viability during and after stress exposure in durum wheat callus. Plant Sci 167:9–18CrossRefGoogle Scholar
  40. Mattos LM, Moretti CL (2015) Oxidative stress in plants under drought conditions and the role of different enzymes. Enzyme Eng 5:1.  https://doi.org/10.4172/2329-6674.1000136 Google Scholar
  41. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410CrossRefGoogle Scholar
  42. Moller IM, Jensen PE, Hansson A (2007) Oxidative modifications to cellular components in plants. Annu Rev Plant Biol 58:459–481CrossRefGoogle Scholar
  43. Monira A, El KA, Naima Z (2012) Evaluation of protective and antioxidant activity of thyme (Thymus vulgaris) extract on paracetamol-induced toxicity in rats. Aust J Basic Appl Sci 6:467–474Google Scholar
  44. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15:473–497CrossRefGoogle Scholar
  45. Nadernejad N, Ahmadimoghadam A, Hosseinifard J, Pourseyedi S (2012) Phenylalanin ammonialyase activity, total phenolic and flavonoid content in flowers, leaves, hulls and kernels of three pistachio (Pistacia vera L.) cultivars. American-Eurasian J Agric Environ Sci 12:807–814Google 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. Patade VY, Bhargava S, Suprasanna P (2012) Effects of NaCl and isosmotic PEG stress on growth, osmolytes accumulation and antioxidant defense in cultured sugarcane cells. Plant Cell Tissue Organ Cult 108:279–286CrossRefGoogle Scholar
  48. Pei ZM, Murata Y, Benning G, Thomine S, Klüsener B, Allen GJ, Grill E, Schroeder JI (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signaling in guard cells. Nature 406:731–734CrossRefGoogle Scholar
  49. Polle A (2001) Dissecting the superoxide dismutase–ascorbate peroxidase–glutathione pathway in chloroplasts by metabolic modeling. Computer simulations as a step towards flux analysis. Plant Physiol 126:445–462CrossRefGoogle Scholar
  50. Putalun W, Luealon W, De-Eknamkul W, Tanaka H, Shoyama Y (2007) Improvement of artemisinin production by chitosan in hairy root cultures of Artemisia annua L. Biotechnol Lett 29:1143–1146CrossRefGoogle Scholar
  51. Ramachandra Rao S, Ravishankar GA (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 20:101–153CrossRefGoogle Scholar
  52. Rao S, Ftz J (2013) In vitro selection and characterization of polyethylene glycol (PEG) tolerant callus lines and regeneration of plantlets from the selected callus lines in sugarcane (Saccharum officinarum L.). Physiol Mol Biol Plants 19:261–268CrossRefGoogle Scholar
  53. Ravishankar GA, Ramachandra Rao S (2000) Biotechnological production of phyto-pharmaceuticals. J Biochem Mol Biol Biophys 4:73–102Google Scholar
  54. Razavizadeh R, Adabavazeh F (2017) Effects of sorbitol on essential oil of Carum copticum L. under in vitro culture. Rom Biotechnol Lett 22:12281–12289Google Scholar
  55. Ronald SF, Laima SK (1999) Phenolics and cold tolerance of Brassica napus. Plant Agric 1:1–5Google Scholar
  56. Saleh L, Plieth C (2009) Fingerprinting antioxidative activities in plants. Plant Methods 5:2CrossRefGoogle Scholar
  57. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Argentine J, Irwin N, Janssen KA, Curtis S, Zierler M, McInerny N, Brown D, Schaefer S (eds) Cold Spring Harbor Laboratory Press, New York, NY pp A1–5Google Scholar
  58. Sandhya V, Ali SKZ, Grover M, Reddy G, Venkateswaralu B (2010) Effect of plant growth promoting Pseudomonas spp. on compatible solutes antioxidant status and plant growth of maize under drought stress. Plant Growth Regul 62:21–30CrossRefGoogle Scholar
  59. Scheumann V, Schoch S, Rüdiger W (1999) Chlorophyll b reduction during senescence of barley seedlings. Planta 209:364–370CrossRefGoogle Scholar
  60. Shao HB, Chu LY, Abdul Jaleel C, Zhao CX (2008) Water-deficit stress-induced anatomical changes in higher plants. C R Biol 331:215–225CrossRefGoogle Scholar
  61. Shao HB, Chu LY, Jaleel CA, Manivannan P, Panneerselvam R, Shao MA (2009) Understanding water deficit stress-induced changes in the basic metabolism of higher plants—biotechnologically and sustainably improving agriculture and the eco environment in arid regions of the globe. Crit Rev Biotechnol 29:131–151CrossRefGoogle Scholar
  62. Sharma P, Dubey RS (2007) Involvement of oxidative stress and role of antioxidative defense system in growing rice seedling exposed to toxic concentration of aluminum. Plant Cell Rep 26:2027–2038CrossRefGoogle Scholar
  63. Sharma P, Jha AB, Dubey RS, Pessarakli M (2012) Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Aust J Bot 2012:1–26Google Scholar
  64. Singh M, Ganesha-Rao RS, Ramesh S (1997) Irrigation and nitrogen requirement of lemongrass (Cymbopogon flexuosus (Sleud) Wats) on a red sandy loam soil under semiarid tropical conditions. J Essent Oil Res 9:569–574CrossRefGoogle Scholar
  65. Sircelj H, Tausz M, Grill D, Batic F (2007) Detecting different levels of drought stress in apple trees (Malus domestica Borkh.) with selected biochemical and physiological parameters. Scientia Hort 113:362–369CrossRefGoogle Scholar
  66. Smetanska I (2008) Production of secondary metabolites using plant cell cultures. Adv Biochem Eng Biotechnol 111:187–228Google Scholar
  67. Somogyi-Nelson M (1952) Notes on sugar determination. J Biol Chem 195:19–29Google Scholar
  68. Sun J, Gu J, Zeng J, Han S, Song AP, Chen FD, Fang WM, Jiang JF, Chen SM (2013) Changes in leaf morphology, antioxidant activity and photosynthesis capacity in two different drought-tolerant cultivars of chrysanthemum during and after water stress. Sci Hortic 161:249–258CrossRefGoogle Scholar
  69. Tátrai ZA, Sanoubar R, Pluhár Z, Mancarella S, Orsini F, Gianquinto G (2016) Morphological and physiological plant responses to drought stress in Thymus citriodorus. Int J Agron 2016:1–8CrossRefGoogle Scholar
  70. Ti-Da GE, SUI F-G, Ping BA (2006) Effects of water stress on the protective enzymes and lipid peroxidation in roots and leaves of summer maize. Agric Sci China 5:291–298CrossRefGoogle Scholar
  71. Wagner GJ (1979) Content and vacuole/extra vacuole distribution of neutral sugars, free amino acids and anthocyanins in protoplast. Plant Physiol 64:88–93CrossRefGoogle Scholar
  72. Willekens H, Chamnongpol S, Davey M, Schraudner M, Langebartels C, Van Montagu M, Inzé D, Van Camp W (1997) Catalase is a sink for H2O2 and is indispensable for stress defence in C-3 plants. EMBO J 16:4806–4816CrossRefGoogle Scholar
  73. Xie SX, Zhang QM, Xiong XY, Carol L (2005) Effect of water-deficit stress on plant gene expression. J Hunan Agric Univ (Natural Sci) 31:574–579Google Scholar
  74. Yaser F, Uzal O, Ozpay T (2010) Changes of lipid peroxidation and chlorophyll amount of green bean genotypes under drought stress. Afr J Agric Res 5:2705–2709Google Scholar
  75. Zahir A, Abbasi BH, Adil M, Anjum S, Zia M, Ihsan-Ul-Haq (2014) Synergistic effects of drought stress and photoperiods on phenology and secondary metabolism of Silybum marianum. Appl Biochem Biotechnol 174:693–707CrossRefGoogle Scholar
  76. Zhuang H, Du J, Wang Y (2011) Antioxidant capacity changes of 3 cultivar Chinese pomegranate (Punica granatum L.) juices and corresponding wines. J Food Sci 76(4):C606–C611.  https://doi.org/10.1111/j.1750-3841.2011.02149.x CrossRefGoogle Scholar
  77. Zou JJ, Wei FJ, Wang C, Wu JJ, Ratnasekera D, Liu WX, Wu WH (2010) Arabidopsis calcium-dependent protein kinase CPK10 functions in abscisic acid- and Ca2+-mediated stomatal regulation in response to drought stress. Plant Physiol 154:1232–1243CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Roya Razavizadeh
    • 1
  • Farnaz Farahzadianpoor
    • 1
  • Fatemeh Adabavazeh
    • 1
  • Setsuko Komatsu
    • 2
    Email author
  1. 1.Department of BiologyPayame Noor UniversityTehranIran
  2. 2.Faculty of Environmental and Information SciencesFukui University of TechnologyFukuiJapan

Personalised recommendations